<p><strong>BACKGROUND: </strong>Studies have demonstrated reduced rotavirus vaccine effectiveness (VE) in resource-limited settings. Enteropathogen co-infections in rotavirus cases have been hypothesized to contribute to the lower vaccine effectiveness in such settings. We sought to determine if co-infections affect rotavirus VE in Botswana.</p>

<p><strong>METHODS: </strong>Between June 2013 and April 2015, children &lt;60 months old, presenting with severe gastroenteritis at four hospitals as part of a national rotavirus surveillance were enrolled. Rotavirus EIA positive samples were tested with an in-house real-time polymerase chain reaction (PCR) panel that detected nine pathogens and a commercial 15 multiplex PCR gastrointestinal pathogen panel (GPP). Co-infection was defined as detection of rotavirus plus one of the five pathogens with the highest attributable fractions for diarrhea. Vaccine status was compared between rotavirus case patients and non-rotavirus "test-negative" controls. Vaccine effectiveness was also calculated restricting cases to those with rotavirus as the only pathogen detected.</p>

<p><strong>RESULTS: </strong>242 children tested rotavirus EIA positive and 368 children were negative. Of the 182 rotavirus EIA-positive samples tested with the GPP assay, co-infections were detected in60 (33%). The overall adjusted 2-dose VE was 59% (95% CI 27-77) in the rotavirus co-infection group and 51% (95% CI -14-79) in the rotavirus mono-infection subgroup. Using in-house multiplex PCR panel, out of 213 rotavirus EIA positive subjects, co-infections were detected in 98 samples (46%). The overall adjusted VE for two doses was 48% (95% CI -2-74) and 62% (95% CI 25-80) in rotavirus mono-infection subgroup.</p>

<p><strong>CONCLUSIONS: </strong>We could not find evidence of an effect of enteric co-infections on the effectiveness of rotavirus vaccine.</p>