Abstract
Broad-range amplification and sequencing of the 16S rRNA gene, directly from clinical samples, is a method that potentially allows detection of any cultivable or non-cultivable bacteria. However, the method is prone to false positive results due to PCR contamination. Another concern is the human DNA abundance compared to bacterial DNA in samples from sterile sites. Those factors may decrease the sensitivity and specificity of the assay and can complicate the analysis and interpretation of the results. The objective of this prospective study was to try to avoid the most common pitfalls, mentioned above, and develop a molecular 16S assay with a high clinical sensitivity and specificity. Fifty-six consecutive tissue samples from patients with suspected deep infections were extracted by 3 different DNA-extraction methods; two based on a principle of bacterial DNA enrichment, and one conventional DNA extraction method. We compared three primer pairs, including both conventional and DPO principle, targeting different variable regions of the 16S rRNA gene. Results from routine tissue culture were used as reference. Clinical data was recorded from patient charts and analyzed in parallel. Of a total of 56 samples, collected from 39 patients, 70% (39 samples) were assessed as true infections by analysis of clinical data. Bacterial enrichment extraction increased sensitivity from 54% to 72%. The 2 sets of primer pairs defining region V1-V3 and V3-V4, showed similar sensitivity, but DPO-primers resulted in better specificity, i.e. less contaminations. The primer pairs covering V1-V8 show significantly lower sensitivity (p < .001) than V1-V3 and V3-V4. Optimizing extraction protocols and choice of primers can increase the sensitivity and specificity of a molecular 16S-analysis, rendering a valuable complement to tissue culture.
Highlights
Deep infections such as abscesses and bone and joint infections can be caused by a wide variety of pathogens and are a significant cause of morbidity and mortality (Mancini et al, 2010; Johansson et al, 2010; Zimmerli et al, 2004)
When comparing the three different DNA extracting methods we obtained the following results from the samples grouped as true infection: with DNA tissue kit from Qiagen (EZ); 54% were positive, and, while using the DNA extraction assays based on the principle of bacterial selective isolation; Ultra-Deep Microbiome Prep (DP), 72% resulted in an identifiable bacterium
The 16S rRNA gene exists in all bacteria, often in multiple copies (Kang et al, 2010), which may improve the sensitivity of molecular detection of 16S, a quality that is desirable in this kind of analysis as the infecting microbes sometimes are present in very small amounts in the sample (Kellogg et al, 2000; Phillips and Bradley, 1990; Wain et al, 1998)
Summary
Deep infections such as abscesses and bone and joint infections can be caused by a wide variety of pathogens and are a significant cause of morbidity and mortality (Mancini et al, 2010; Johansson et al, 2010; Zimmerli et al, 2004). Today the gold standard method to detect bacteria in deep infections is culture-based diagnostics. Antibiotic treatment may kill the bacteria or render them too weak to give rise to visible colonies In these cases it is desirable to have a method complementary to the culture diagnostics. A frequently used method in parallel with culture diagnostics is amplification and Sanger sequencing of the 16S rRNA gene. This method potentially allows detection of any cultivable or non-cultivable bacteria (Chakravorty et al, 2015; Drancourt et al, 2004; Bosshard et al, 2003; Bosshard et al, 2004; Woo et al, 2008)
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