Etiology of Acute Febrile Illness in the Peruvian Amazon as determined by modular formatted quantitative PCR: A Protocol for RIVERA, a Health Facility-Based Case-Control Study.

Background: The study of the etiology of acute febrile illness (AFI) has historically been designed as a prevalence of pathogens detected from a case series. This strategy has an inherent unrealistic assumption that all pathogen detection allows for causal attribution, despite known asymptomatic carriage of the principal causes of acute febrile illness in most low- and middle-income countries (LMICs). We designed a semi-quantitative PCR in a modular format to detect bloodborne agents of acute febrile illness that encompassed common etiologies of AFI in the region, etiologies of recent epidemics, etiologies that require an immediate public health response and additional pathogens of unknown endemicity. We then designed a study that would delineate background levels of transmission in the community in the absence of symptoms to provide corrected estimates of attribution for the principal determinants of AFI. Methods: A case-control study of acute febrile illness in patients ten years or older seeking health care in Iquitos, Loreto, Peru, was planned. Upon enrollment, we will obtain blood, saliva, and mid-turbinate nasal swabs at enrollment with a follow-up visit on day 21-28 following enrollment to attain vital status and convalescent saliva and blood samples, as well as a questionnaire including clinical, socio-demographic, occupational, travel, and animal contact information for each participant. Whole blood samples are to be simultaneously tested for 32 pathogens using TaqMan array cards. Mid-turbinate samples will be tested for SARS-CoV-2, Influenza A and Influenza B. Conditional logistic regression models will be fitted treating case/control status as the outcome and with pathogen-specific sample positivity as predictors to attain estimates of attributable pathogen fractions for AFI. Discussion: The modular PCR platforms will allow for reporting of all primary results of respiratory samples within 72 hours and blood samples within one week, allowing for results to influence local medical practice and enable timely public health responses. The inclusion of controls will allow for a more accurate estimate of the importance of specific, prevalent pathogens as a cause of acute illness. Study Registration: Project 1791, Registro de Proyectos de Investigación en Salud Pública (PRISA), Instituto Nacional de Salud, Perú.

"Candidatus Campylobacter infans" detection is not associated with diarrhea in children under the age of 2 in Peru.

A working hypothesis is that less common species of Campylobacter (other than C. jejuni and C. coli) play a role in enteric disease among children in low resource settings and explain the gap between the detection of Campylobacter using culture and culture independent methods. "Candidatus Campylobacter infans" (C. infans), was recently detected in stool samples from children and hypothesized to play a role in Campylobacter epidemiology in low- and middle-income countries (LMIC). This study determined the prevalence of C. infans in symptomatic and asymptomatic stool samples from children living in Iquitos, Peru. Stool samples from 215 children with diarrhea and 50 stool samples from children without diarrhea under the age of two were evaluated using a multiplex qPCR assay to detect Campylobacter spp. (16S rRNA), Campylobacter jejuni / Campylobacter coli (cadF gene), C. infans (lpxA), and Shigella spp. (ipaH). C. infans was detected in 7.9% (17/215) symptomatic samples and 4.0% (2/50) asymptomatic samples. The association between diarrhea and the presence of these targets was evaluated using univariate logistic regressions. C. infans was not associated with diarrhea. Fifty-one percent (75/146) of Campylobacter positive fecal samples were negative for C. jejuni, C. coli, and C. infans via qPCR. Shotgun metagenomics confirmed the presence of C. infans among 13 out of 14 positive C. infans positive stool samples. C infans explained only 20.7% of the diagnostic gap in stools from children with diarrhea and 16.7% of the gap in children without diarrhea. We posit that poor cadF primer performance better explains the observed gap than the prevalence of atypical non-C. jejuni/coli species.

Shotgun metagenomics of fecal samples from children in Peru reveals frequent complex co-infections with multiple Campylobacter species.

Campylobacter spp. are a major cause of bacterial diarrhea worldwide and are associated with high rates of mortality and linear growth faltering in children living in low- to middle-income countries (LMICs). Campylobacter jejuni and Campylobacter coli are most often the causative agents of enteric disease among children in LMICs. However, previous work on a collection of stool samples from children under 2 years of age, living in a low resource community in Peru with either acute diarrheal disease or asymptomatic, were found to be qPCR positive for Campylobacter species but qPCR negative for C. jejuni and C. coli. The goal of this study was to determine if whole-genome shotgun metagenomic sequencing (WSMS) could identify the Campylobacter species within these samples. The Campylobacter species identified in these stool samples included C. jejuni, C. coli, C. upsaliensis, C. concisus, and the potential new species of Campylobacter, "Candidatus Campylobacter infans". Moreover, WSMS results demonstrate that over 65% of the samples represented co-infections with multiple Campylobacter species present in a single stool sample, a novel finding in human populations.