Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

Abstract

The study was performed to compare real‐time PCR after nucleic acid extraction directly from stool samples as well as from samples stored and transported on Whatman papers or flocked swabs at ambient temperature in the tropics. In addition, the possible suitability for a clear determination of likely aetiological relevance of PCR‐based pathogen detections based on cycle threshold (Ct) values was assessed. From 632 Tanzanian children <5 years of age with and without gastrointestinal symptoms, 466 samples were subjected to nucleic acid extraction and real‐time PCR for gastrointestinal viral, bacterial and protozoan pathogens. Equal or even higher frequencies of pathogen detections from Whatman papers or flocked swabs were achieved compared with nucleic acid extraction directly from stool samples. Comparison of the Ct values showed no significant difference according to the nucleic acid extraction strategy. Also, the Ct values did not allow a decision whether a detected pathogen was associated with gastrointestinal symptoms.

Seasonal Differences in Cyclospora cayetanensis Prevalence in Colombian Indigenous People

“…This has been suggested as a hint for lower pathogenicity or for the discrimination between infection and sole colonization [43][44][45]. However, contradicting results from other studies in high-endemicity settings make this assumption controversial [10,46,47].…”

New Developments in PCR-Based Diagnostics for Bacterial Pathogens Causing Gastrointestinal Infections—A Narrative Mini-Review on Challenges in the Tropics

“…In a recently performed “real-life” observation in tropical Tanzania [ 30 ], bacterial DNA in stool samples both on Whatman cards and on nucleic acid-stabilizing swabs remained stable for several months of storage at ambient temperature. Positivity rates of multiplex PCR were comparable after immediate nucleic acid extraction from stool and after delayed nucleic acid extraction from Whatman cards and from swabs.…”

“…Positivity rates of multiplex PCR were comparable after immediate nucleic acid extraction from stool and after delayed nucleic acid extraction from Whatman cards and from swabs. However, imperfect agreement as defined by Cohen’s kappa [ 31 ] was observed [ 30 ], a finding which is not easy to interpret. Differing sensitivity due to parallelism of variable free nucleic acid degradation and variable nucleic acid release from decaying cells is one potential explanation.…”

“…Another likely explanation is release of cross-reacting DNA of non-target organisms after prolonged storage. Accordingly, a residual uncertainty regarding diagnostic accuracy remains [ 30 ].…”

“…When PCR from stool of patients with diarrhea is performed in resource-rich Western industrialized countries like Germany [ 83 ] or Switzerland [ 111 ], even after travelling in the tropics under hygienically controlled circumstances [ 111 , 112 ], only individual pathogens are usually detected that are then causally attributed to a coexisting gastrointestinal disease. Under tropical conditions, however, such etiological attribution is not as easy, because DNA of typical gastrointestinal pathogens such as S. enterica , Campylobacter spp., diarrheagenic E. coli [ 7 , 13 , 14 , 30 ] and even Shigella spp. [ 19 ] or enteropathogenic protozoa [ 7 , 13 , 14 , 18 , 19 , 30 , 113 ] can be detected in asymptomatic individuals in the tropics as well.…”