Elimination of host and dead pathogenic organism DNA for improved diagnostic outcome
Date
2019Author
Mann, B.C.
Grobler, A.F.
Vermeulen, U.
Swanevelder, S.
Metadata
Show full item recordAbstract
Molecular-based diagnostic techniques used to detect bacterial
infections in clinical samples are often unable to discriminate
between living and dead bacteria and extracellular DNA. This
presents a major problem, leading to reduced specificity and
sometimes in a failure to properly monitor the effectiveness of
antimicrobial therapy. An additional problem faced by molecular
diagnostic assays as well as next generations sequencing (NGS)-
based diagnostic approaches is the presence of high quantities of
contaminating host DNA, which often obscures the presence of and
changes in microbial populations due to the majority of sequencing
reads aligning to host DNA. Here we describe the effect of two
buffers used to achieve live/dead differentiation and the removal of
contaminating host cell DNA. We evaluated these buffers in two
different scenarios; one focusing on a previously validated TB
diagnostic and one following a NGS-based approach. For the
diagnostic method, 101 fresh sputum samples were treated with
the live/dead differentiation buffer before lysis and molecular
diagnosis by quantitative polymerase chain reaction (q-PCR). For
future NGS applications, samples will first be treated with a
differential lysis buffer to selectively lyse any host cells present. This
will be followed by treatment with a DNA removal buffer and consequent q-PCR and sequencing. The q-PCR results were evaluated
against Sputum smear microscopy (S), Culture (C) and GeneXpert
MTB/RIF Ultra. Of the 101 samples analysed, 13 were culture
contaminated and 7 were Non-Tuberculosis mycobacteria (NTM). The basic test identified 12 and the live/dead differentiation test
10 samples respectively as positive from the culture negative
samples. A total of 11 samples indicated the presence of DNA from
dead bacilli, where the ΔCt value was more than the threshold value
of 2.5. The molecular test, both with and without live/dead
differentiation, was able to identify 2 positive MTBC samples from
the 13 contaminated cultures. Both the basic and the live/dead
differentiation test, were able to identify 3/4 first time GeneXpert
Ultra samples and had a 100% specificity for NTM. The inclusion of a
live/dead differentiation buffer improves the specificity of the
molecular test but may have a negative effect on the sensitivity
especially on samples with a low bacilli starting concentration. A few
false positives were indicated but could be due to the fact that some
of the culture results were false negative as confirmed by follow-up
tests. Further optimization of the live/dead differentiation process
can enhance the specificity. This includes an increase in the
concentration of the buffer or an initial liquefaction step of the
sputum before proceeding with the live/dead differentiation step.
The inclusion of a live/dead differentiation buffer indicated that DNA
from dead bacilli was present in some of the samples
URI
http://hdl.handle.net/10394/33320https://www.sciencedirect.com/science/article/pii/S1056871919303260
https://doi.org/10.1016/j.vascn.2019.106608Get