Abstract
BackgroundThe detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. We observed that such detection was hampered in diarrheic stool specimens and we set-up an improved protocol combining lyophilization of stools prior to a semi-automated DNA extraction.FindingsA total of 41 human diarrheic stool specimens comprising of 35 specimens negative for enteropathogens and six specimens positive for Salmonella enterica in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA was extracted from lyophilised and non-lyophilised aliquots by combining automatic and phenol-chloroform DNA extraction. DNA was incorporated into real-time PCRs targeting the 16S rRNA gene of Bacteria and the archaea Methanobrevibacter smithii and the chorismate synthase gene of S. enterica. Whereas negative controls consisting in DNA-free water remained negative, M. smithii was detected in 26/41 (63.4%) non-lyophilised (Ct value 28.78 ± 9.1) versus 39/41 (95.1%) lyophilised aliquots (Ct value 22.04 ± 5.5); bacterial 16S rRNA was detected in 33/41 (80.5%) non-lyophilised (Ct value 28.11 ± 5.9) versus 40/41 (97.6%) lyophilised aliquots (Ct value 24.94 ± 6.6); and S. enterica was detected in 6/6 (100%) non-lyophilized and lyophilized aliquots (Ct value 26.98 ± 4.55 and 26.16 ± 4.97, respectively). S. enterica was not detected in the 35 remaining diarrheal-stool specimens. The proportion of positive specimens was significantly higher after lyophilization for the detection of M. smithii (p = 0.00043) and Bacteria (p = 0.015).ConclusionLyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. The semi-automated protocol described here could be routinely used for the molecular diagnosis of infectious diarrhea.
Highlights
The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences
Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms
Infectious diarrhea is caused by a wide spectrum of enteropathogens including the bacteria Salmonella spp., enteropathogenic Escherichia coli, Shigella spp., Yersinia spp., Campylobacter spp. and Clostridium difficile [1] and noroviruses, rotaviruses, toroviruses, coronaviruses, astroviruses, enteroviruses and adenoviruses, all pathogens reportedly causing 50% of cases of diarrhea [2]
Summary
The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. Conclusion: Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. Previous studies showed that a preliminary enrichment step applied to stool increases the detection of enteropathogen DNA [12,13]. We aimed to optimize the DNA extraction protocol to target both bacteria and archaea in diarrheal stool specimens.
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