DNA was extracted using a QIAmp DNA stool mini kit (QIAGEN, Hilden, Germany), following the manufacturer’s protocol. Extracts were thereafter stored at −20 °C. A part of the stool samples was preserved in 5% formalin to perform the microscopic analysis. PCR investigations were carried out on site. In addition, extracts and formalin samples were sent to the Bernhard–Nocht Institute for Tropical Medicine (BNITM, Hamburg, HH, Germany). During the BNITM microscopy, control examinations and further analysis were performed. Airfreight requirements were fulfilled, the cooling chain was not interrupted (based on the temperature control documentation), and transport was done by a specialized company (World Courier, Frankfurt, HE, Germany).
In-house real-time multiplex PCRs for protozoan and helminthic parasites targeting Entamoeba histolytica, Giardia intestinalis, Cryptosporidium spp., and Cyclospora cayetanensis, as well as Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Ancylostoma spp., Trichuris trichiura, Schistosoma spp., Enterobius vermicularis, Taenia saginata, Taenia solium, and Hymenolepis nana were performed as described before [3 (link)]. Further, enteroinvasive bacterial pathogens like Campylobacter jejuni, Salmonella spp., Shigella ssp./enteroinvasive E. coli (EIEC), and Yersinia spp. were assessed by in-house real-time PCR [4 (link)]. To identify diarrheagenic E. coli infections, Rida Gene RT-PCR assays for enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxin-producing E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) (R-Biopharm AG, Darmstadt, HE, Germany) were used, as described before [5 (link)]. Furthermore, a species-specific PCR for Tropheryma whipplei [6 (link)] was applied [7 (link)]. The DNA of phocid herpesvirus was included as an internal control for the in-house PCRs [8 (link),9 (link)].
In all RT-PCRs runs, positive and negative controls were included: As positive controls, synthetically designed target sequences linked by EcoR1 endonuclease restriction sites and inserted into pEX-A128 vector backbones were used (Eurofins Scientific SE). Negative controls included PCT water samples that had undergone the whole nucleic acid extraction process to exclude sample contamination. All primers and probes of the in-house PCRs were purchased from Eurofins, Hamburg, HH, Germany. The assays were performed on a multi-channel RotorGene Q Cycler (Qiagen, Hilden, HE, Germany).
In addition, light microscopy of all stool samples by direct saline and/or iodine mounts and following a formol-ethyl acetate concentration was performed.
In-house real-time multiplex PCRs for protozoan and helminthic parasites targeting Entamoeba histolytica, Giardia intestinalis, Cryptosporidium spp., and Cyclospora cayetanensis, as well as Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Ancylostoma spp., Trichuris trichiura, Schistosoma spp., Enterobius vermicularis, Taenia saginata, Taenia solium, and Hymenolepis nana were performed as described before [3 (link)]. Further, enteroinvasive bacterial pathogens like Campylobacter jejuni, Salmonella spp., Shigella ssp./enteroinvasive E. coli (EIEC), and Yersinia spp. were assessed by in-house real-time PCR [4 (link)]. To identify diarrheagenic E. coli infections, Rida Gene RT-PCR assays for enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxin-producing E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) (R-Biopharm AG, Darmstadt, HE, Germany) were used, as described before [5 (link)]. Furthermore, a species-specific PCR for Tropheryma whipplei [6 (link)] was applied [7 (link)]. The DNA of phocid herpesvirus was included as an internal control for the in-house PCRs [8 (link),9 (link)].
In all RT-PCRs runs, positive and negative controls were included: As positive controls, synthetically designed target sequences linked by EcoR1 endonuclease restriction sites and inserted into pEX-A128 vector backbones were used (Eurofins Scientific SE). Negative controls included PCT water samples that had undergone the whole nucleic acid extraction process to exclude sample contamination. All primers and probes of the in-house PCRs were purchased from Eurofins, Hamburg, HH, Germany. The assays were performed on a multi-channel RotorGene Q Cycler (Qiagen, Hilden, HE, Germany).
In addition, light microscopy of all stool samples by direct saline and/or iodine mounts and following a formol-ethyl acetate concentration was performed.
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