Validation of microbial source tracking markers for the attribution of fecal contamination in indoor-household environments of the Peruvian Amazon

Abstract

The performance of eight microbial source tracking (MST) markers was evaluated in a low-resource, tropical community located in Iquitos, Peru. Fecal samples from humans, dogs, cats, rats, goats, buffalos, guinea-pigs, chickens, ducks, pigeons, and parrots were collected ( n = 117). All samples were tested with human ( BacHum, HF183-Taqman ), dog ( BactCan ), pig ( Pig-2-Bac ), and avian ( LA35, Av4143, ND5, cytB ) markers using quantitative PCR (qPCR). Internal validity metrics were calculated using all animal fecal samples, as well as animal fecal samples contextually relevant for the Peruvian Amazon. Overall, Pig-2-Bac performed best, with 100% sensitivity and 88.5% specificity to detect the correct fecal source. Human-associated markers showed a sensitivity of 80.0% and 76.7%, and specificity of 66.2% and 67.6%. When limiting the analysis to contextually relevant animal fecal samples for the Peruvian Amazon, Av143 surpassed cytB with 95.7% sensitivity and 81.8% specificity. BactCan demonstrated 100% sensitivity and 47.4% specificity. The gene copy number detected by BacHum and HF183-Taqman were positively correlated (Pearson's correlation coefficient: 0.785), as well as avian markers cytB with Av4143 (Pearson's correlation coefficient: 0.508) and nd5 (Pearson's correlation coefficient: 0.949). These findings suggest that markers such as Av4143, Pig2Bac, cytb and BacHum have acceptable performance to be impactful in source attribution studies for zoonotic enteric disease transmission in this and similar low-resource communities.

Associations among Household Animal Ownership, Infrastructure, and Hygiene Characteristics with Source Attribution of Household Fecal Contamination in Peri-Urban Communities of Iquitos, Peru

“…The absence of positive LA35 markers is attributed to the particularly low sensitivity of this marker. 29 Limitations of this study include the fact that the performance of MST markers is setting specific and requires a previous validation step. In addition, new human MST markers that have higher degrees of sensitivity and specificities are required.…”

“…Eight MST markers that have previously been validated within this context were used to score surface samples. 29 Specifically, two avian fecal markers (Av4143 and LA35), two avian mitochondrial fecal markers (cytb and ND5), two human fecal markers (BachHum and HF83-Taqman), one dog fecal marker (BactCan), and one pig fecal marker (Pig2Bac) were used. [33][34][35][36][37] Details regarding the target species, gene, and internal validity parameters of all eight MST markers are presented in Supplemental Table 1.…”

“…Standard curve analysis was performed as reported previously. 29 Detection of Campylobacter spp. Surface samples were tested for the presence or absence of Campylobacter spp.…”

“…Determination of assayspecific limits of detection has been described previously. 29 Floor and table surface samples were treated as distinct independent samples. Floor was categorized as either finished-made of a material that separated the dirt floor from household members or animals-or unfinished-uncovered earth.…”

“…[25][26][27][28] Using MST markers previously validated in the same region, this study quantified the burden of animal fecal contamination of surfaces by animal species, in households in a peri-urban, low-resource, tropical community of Loreto, Peru, with a particular emphasis on avian fecal contamination. 29 We assessed the associations between MST presence and burden in household surfaces with household infrastructure and socioeconomic characteristics of the primary caregiver. Finally, we assess the association between the detection of Campylobacter spp.…”