ABSTRACT
Several bacteria, viruses, and parasites cause diarrhea as coinfecting pathogens. We designed a DNA microarray comprising 60-bp probes spotted 194 times for the multiplex detection of 33 enteropathogenic bacteria and seven enteropathogenic viruses, and the archaeon Methanobrevibacter smithii was used as an internal positive control. Nine pathogen-free stool specimens were used as negative controls. One of these control specimens was further spiked with Salmonella enterica as a positive control. The microarray was then tested with 40 pathological stool specimens, comprising S. enterica (n = 30), Campylobacter jejuni (n = 4), pathogenic Escherichia coli (n = 2), and adenovirus (n = 4). M. smithii was detected in 47/49 (95.9%) specimens, no pathogen was detected in negative controls and S. enterica was identified in the S. enterica-spiked positive control. The overall specificity was 100% and the overall sensitivity was 97.5% because one S. enterica sample was missed by the microarray. The multiplexed detection of C. jejuni spiked into an adenovirus-positive stool sample gave positive results, with fluorescence values of 14.3 and 9.1, respectively. These data indicate that using the protocol developed in this article, the DNA array allows for the multiplexed detection of some enteropathogens in stool samples.
Subject(s)
Diarrhea/diagnosis , Enterobacteriaceae Infections/diagnosis , Oligonucleotide Array Sequence Analysis/methods , Salmonella enterica/isolation & purification , Adenoviridae/isolation & purification , Adenoviridae Infections/diagnosis , Campylobacter jejuni/isolation & purification , DNA, Bacterial/isolation & purification , DNA, Viral/isolation & purification , Diarrhea/microbiology , Diarrhea/virology , Feces/microbiology , Feces/virology , Genes, Bacterial , Humans , Sensitivity and SpecificityABSTRACT
BACKGROUND: The 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. FINDINGS: A 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). CONCLUSION: Lyophilization 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.