Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples

Menu, Estelle; Mary, Charles; Toga, I.; Raoult, Didier; Ranque, Stéphane; Bittar, Fadi

doi:10.1186/s13104-018-3300-2

Cited by 48 publications

(35 citation statements)

References 14 publications

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“…The automated procedure using the EZ1 Advanced XL (Qiagen, Hilden, German) with the DNA Tissue Kit (Qiagen) according to the manufacturer's recommendation [8]. The RNA were extracted from 200 μL clear supernatant using the EZ1 Advanced XL (Qiagen, Hilden, German) with the Virus Mini Kit v2.0 (Qiagen) according to the manufacturer's recommendation.…”

Section: Gastrointestinal Specimensmentioning

confidence: 99%

Respiratory and gastrointestinal infections at the 2017 Grand Magal de Touba, Senegal: A prospective cohort survey

Hoang

Goumballa

Dao

et al. 2019

Travel Medicine and Infectious Disease

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Background: The Grand Magal of Touba is the largest Muslim pilgrimage in Senegal with a potential for infectious disease transmission. Methods: Clinical follow-up, adherence to preventive measures and qPCR-based respiratory and gastrointestinal pathogens carriage pre-and post-Magal, were assessed. Results: 110 pilgrims from South Senegal were included. The duration of stay in Touba was 3 days. 41.8% and 14.5% pilgrims reported respiratory and gastrointestinal symptoms. Most individuals having the onset of symptoms during their stay in Touba, or soon after returning. The acquisition of rhinoviruses, coronaviruses and adenovirus was 13.0, 16.7 and 4.6% respectively and that of Streptococcus pneumoniae and Haemophilus influenzae was 3.7% and 26.9%.Acquisition of gastrointestinal viruses and parasites was low, while bacterial acquisition ranged from 2.2% for Campylobacter jejuni to 33.0% for enteropathogenic Escherichia coli. Conclusion: This preliminary study confirms that Grand Magal pilgrims are likely to be exposed to communicable disease risk as observed in other pilgrimage settings. Further study including larger numbers of pilgrims are needed to investigate potential risk factors for respiratory and gastrointestinal infections at the Grand Magal.

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Section: Gastrointestinal Specimensmentioning

confidence: 99%

Respiratory and gastrointestinal infections at the 2017 Grand Magal de Touba, Senegal: A prospective cohort survey

Hoang

Goumballa

Dao

et al. 2019

Travel Medicine and Infectious Disease

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“…The total stool DNA was extracted with the EZ1 DNA tissue kit (Qiagen GmbH, Hilden, Germany) according to the procedure described herein [28]. Briefly, 200 mg (200 µL if liquid stools) and 350 µL of G2 lysis buffer were added in a 1.5 mL tube containing 200 mg of 2 mm glass powder.…”

Section: Dna Extractionmentioning

confidence: 99%

Blastocystis Colonization Is Associated with Increased Diversity and Altered Gut Bacterial Communities in Healthy Malian Children

et al. 2019

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Blastocystis is the most common protozoan colonizing the gut of vertebrates. It modulates the human digestive microbiota in the absence of inflammation and gastrointestinal disease. Although it has been associated with human diseases, including inflammatory bowel disease, its pathogenicity remains controversial. This study aimed to assess the influence of Blastocystis on the gut bacterial communities in healthy children. We conducted a cross-sectional study on 147 Blastocystis-colonized and 149 Blastocystis-noncolonized Malian children, with Blastocystis colonization assessed by real-time PCR and gut microbial communities characterized via 16S rRNA gene (Illumina MiSeq) sequencing and bioinformatics analysis. The gut microbiota diversity was higher in Blastocystis-colonized compared to Blastocystis-noncolonized children. The phyla Firmicutes, Elusimicrobia, Lentisphaerae, and Euryarchaeota were higher in Blastocystis-colonized children, whereas Actinobacteria, Proteobacteria, unassigned bacteria, and Deinococcus–Thermus were higher in Blastocystis-noncolonized children. Moreover, Faecalibacterium prausnitzii (family Ruminococcaceae) and Roseburia sp. (family Lachnospiraceae) abundance was higher in Blastocystis-colonized children. We conclude that Blastocystis colonization is significantly associated with a higher diversity of the gut bacterial communities in healthy children, while it is not associated with the presence of potentially pathogenic bacteria in the human gut.

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“…Longer time of fixation may impact the diagnostic accuracy, but this has not been investigated in the present study. Lysis and extraction of DNA are important and can influence the results [39,40,54]. However, the method in this study showed no statistically significant difference in reproducibility between fixed and unpreserved samples.…”

Section: Discussionmentioning

confidence: 58%

“…The most common gastrointestinal parasites included in NAbased diagnostics in Sweden are E. histolytica, G. intestinalis and Cryptosporidium spp. Different cell lysis methods have been used prior to protozoan DNA extraction to minimize inhibitors of the PCR process [32,33] and to disrupt robust cell walls [34][35][36][37][38][39][40]. A reduced yield of parasite-DNA has been reported from fixed samples [41,42].…”

Section: Introduction/backgroundmentioning

confidence: 99%

Optimization of routine microscopic and molecular detection of parasitic protozoa in SAF-fixed faecal samples in Sweden

Ögren

Dienus

Matussek

2019

Infectious Diseases

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Background: Nucleic acid-based methods are increasingly used for screening of gastrointestinal parasites. Microscopy is still used and Swedish routine protocol consists of formalin ethyl-acetate concentration and do not include screening for trophozoites or Cryptosporidium spp. This study aimed to compare detection with the Swedish routine microscopy method to an extended method that includes screening for trophozoites and Cryptosporidium. Furthermore, we also developed a method for DNA recovery from SAF-fixed faecal samples and compared the real-time PCR detection of Giardia intestinalis, Dientamoeba fragilis, Cryptosporidium spp., Entamoeba histolytica and Entamoeba dispar from SAF-fixed and unpreserved faecal samples. PCR results were then compared with microscopy results. Methods: SAF-fixed and unpreserved faecal samples from 1000 patients at the Clinical microbiology laboratory in Region J€ onk€ oping County, Sweden, were included. Samples were analysed with routine formalin ethyl-acetate concentration, wet mounts from both concentrated and unconcentrated samples, Ziehl-Neelsen staining on patients with certain symptoms and real-time PCR. Results: We found a significant higher detection rate of parasites with the extended microscopy method compared to the Swedish routine microscopy method when SAF-fixed samples were used. The detection rate with real-time PCR in SAF-fixed samples was equal to the detection rate in unpreserved samples. There was no significant difference in detection comparing extended microscopy and real-time PCR. Conclusion: In conclusion, this study showed that the extended microscopy method increased detection of intestinal protozoa with detection of both trophozoites and Cryptosporidium spp. We also showed that SAF-fixative can be used for detection of parasite-DNA with real-time PCR.

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Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples

Cited by 48 publications

References 14 publications

Respiratory and gastrointestinal infections at the 2017 Grand Magal de Touba, Senegal: A prospective cohort survey

Respiratory and gastrointestinal infections at the 2017 Grand Magal de Touba, Senegal: A prospective cohort survey

Blastocystis Colonization Is Associated with Increased Diversity and Altered Gut Bacterial Communities in Healthy Malian Children

Optimization of routine microscopic and molecular detection of parasitic protozoa in SAF-fixed faecal samples in Sweden

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