Man with tarry stools.
Ontology highlight
ABSTRACT:
SUBMITTER: Tsai HB
PROVIDER: S-EPMC8709810 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:BACKGROUND: The PCR-based detection of archaea DNA in human specimens relies on efficient DNA extraction. We previously designed one such protocol involving only manual steps. In an effort to reduce the workload involved, we compared this manual protocol to semi-automated and automated protocols for archaea DNA extraction from human specimens. FINDINGS: We tested 110 human stool specimens using each protocol. An automated protocol using the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card and the EZ1® DNA Tissue Kit (Qiagen, Courtaboeuf, France) yielded 35/110 (32%) positives for the real-time PCR detection of the Methanobrevibacter smithii 16S rRNA gene, with average Ct values of 36.1. A semi-automated protocol combining glass-powder crushing, overnight proteinase K digestion and lysis in the buffer from the EZ1 kit yielded 90/110 (82%) positive specimens (P?=?0.001) with an average Ct value of 27.4 (P?=?0.001). The manual protocol yielded 100/110 (91%) positive specimens (P?=?0.001) with an average Ct value of 30.33 (P?=?0.001). However, neither the number of positive specimens nor the Ct values were significantly different between the manual protocol and the semi-automated protocol (P?>?0.1 and P?>?0.1). CONCLUSION: Proteinase K digestion and glass powder crushing dramatically increase the extraction yield of archaea DNA from human stools. The semi-automated protocol described here was more rapid than the manual protocol and yielded significantly more archaeal DNA. It could be applied for extracting total stool DNA for further PCR amplification.
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Project description:BackgroundBloody stools in a neonate may stand for a spectrum of conditions ranging from benign to life-threatening. It is critical to detect the cases that have significant underlying pathology, especially those which require urgent surgical intervention. Previous studies always focused on one particular disease related to bloody stools in neonates, or the study only involved a small number of cases. This study aimed to investigate the common causes of bloody stools in neonates.MethodsThis retrospective cohort study included the neonates admitted to our institution due to "bloody stools" over a 5-year period. We compared the differences among patients' characteristics, feeding choice, underlying diseases, and operation rate between preterm and term neonates.ResultsA total of 300 patients were included, accounting for 1.1% of the total neonatal admissions. The overall rate of exclusive breastfeeding was 28.0%. The most common underlying causes for bloody stools were: cow's milk protein allergy (CMPA, 53.3%), swallowed blood syndrome (10.0%), viral enteritis (9.7%), necrotizing enterocolitis (NEC) > stage II (8.3%), non-specific enteritis (7.3%), and anal fissure (5.0%). The median [interquartile range (IQR)] onset age for bloody stools for all infants was 12 [3-22] days after birth. Preterm neonates had a lower rate of exclusive breastfeeding (P=0.844), higher incidence of NEC > stage II (P=0.014), later bloody stools onset age (P<0.001), and longer length of hospital stay than term neonates (P<0.001). For neonates with NEC, those with bottle-fed had an earlier onset age for bloody stools than those with breast-fed (P=0.027). Only 1.7% (n=5) required surgery (2 stage III NEC, 1 post-NEC stricture, and 2 volvuli). Survival at hospital discharge was 100%.ConclusionsBloody stools in neonates is generally a benign, self-limiting disorder, not related to surgical conditions. The overall operation rate among neonates with bloody stools was only 1.7%. CMPA and NEC were the most common underlying non-surgical and surgical diseases, respectively, for neonates with bloody stools. Feeding choice is related to bloody stools in neonates, policies and strategies to support breastfeeding should be strengthened in the future.
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