Seasonal Influenza A Virus in Feces of Hospitalized Adults

In a cohort of hospitalized adults with seasonal influenza A in Hong Kong, viral RNA was frequently (47%) detected in stool specimens. Viable virus was rarely isolated. Viral RNA positivity had little correlation with gastrointestinal symptoms and outcomes. In vitro studies suggested low potential for seasonal influenza viruses to cause direct intestinal infections.

RNA detection during hospitalization, regardless of gastrointestinal symptoms. Clinical information was prospectively recorded (8). For comparison, fecal shedding of respiratory syncytial virus (RSV) and parainfl uenza virus (PIV) were studied during a 10-month period by using a similar approach. Ethical approval for the study was obtained from the institutional review boards of The Chinese University of Hong Kong.
All stool specimens were subjected to infl uenza viral RNA detection by using quantitative real-time reverse transcription PCR targeting the matrix gene (6). If positive, virus subtyping was performed by using H1-and H3specifi c conventional PCRs. Freshly collected stool specimens during 1 seasonal peak were simultaneously subjected to viral RNA detection and virus isolation by using MDCK cells. Detailed methods for fecal detection of infl uenza viruses and RSV and PIV are provided in the online Technical Appendix (wwwnc.cdc.gov/EID/pdfs/11-0205-Techapp.pdf).
Lectin histochemical analysis and double immunofl uorescence staining were used to study the distribution of infl uenza virus receptors on human small and large intestinal tissues. An in vitro virus binding study on intestinal tissues was also performed by using inactivated human virus isolates of subtypes H1N1 (A/HongKong/ CUHK-13003/2002) and H3N2 (A/HongKong/CUHK-22910/2004) (online Technical Appendix).
A total of 119 hospitalized adults with seasonal infl uenza A infections were studied (Table 1). Their median age was 71 years (interquartile range [IQR] 57-79 years), and most (66%) had concurrent conditions; ≈5% were profoundly immunosuppressed. Vomiting and diarrhea were reported by 15 (13%) and 7 (6%) patients, respectively. Infl uenza A viral RNA was detected in 56 of 119 of stool samples, collected at a median interval of 3 days (IQR 3-5 days) from onset (detection rates by study year and virus subtype are shown in Table 2). Detection rate by day from onset ranged from 31% to 63% and showed a trend to decrease toward the end of the week (Figure 1, panel A). Overall, the mean ± SD fecal viral RNA concentration was 4.4 ± 0.8 log 10 copies/g of feces and the median (IQR) was 4.2 (3.8-5.0) log 10 copies/g of feces; concentrations tended to decrease with longer time elapsed from onset ( Figure 1, panel B).
In most (77%) viral RNA-positive samples, further H1-or H3-specifi c PCRs identifi ed 7 cases as H1 and 36 cases as H3; unsuccessful amplifi cation was associated with lower viral (matrix gene) concentrations (median ] log 10 copies/g of stool; p = 0.04). No discrepancy was found between these and the subtyping results of the virus isolates from NPAs. Fecal viral RNA detection rate and concentrations were similar between H1 and H3 subtypes ( Table 2).
Thirty-eight stool samples from 1 seasonal peak were subjected to virus isolation and viral RNA detection. In 10 cases, cytotoxicity occurred (procedure discontinued); in the remaining 28 cases, 12 were viral RNA positive; only 1 showed virus growth. This sample was from an 82-year-old man with dilated cardiomyopathy hospitalized for seasonal infl uenza A (H1N1) pneumonia and heart failure; diarrhea was absent.
Among Patients with positive and negative fecal viral RNA detection results were compared (Table 1). Positive fecal viral RNA detection was associated with younger age, shorter interval from illness onset to sample collection, lymphopenia, and positive virus isolation. Multivariate logistic regression showed that lymphopenia (adjusted odds ratio 2.36, 95% confi dence interval 1.02-5.47; p = 0.045) and positive virus isolation in NPAs (adjusted odds ratio 3.76, 95% confi dence interval 1.07-13.20; p = 0.039) were signifi cant explanatory variables. No signifi cant association was found between fecal viral RNA detection and clinical outcomes. Fecal viral RNA concentrations were also analyzed, and no association with clinical outcomes was found (data not shown), except for a negative correlation with lymphocyte count (Spearman ρ −0.37, p = 0.047).
Lectin histochemical analysis showed that sialic acid α 2,6-Gal (human-like infl uenza virus receptor) was absent from epithelial surface of small and large intestines and was found only in lamina propria cells. In contrast, sialic acid α 2,3-Gal (avian-like infl uenza virus receptor) was found on large intestinal epithelial cells and in lamina propria cells. Virus-binding study showed that neither seasonal infl uenza A (H1N1) nor A (H3N2) virus bind to small and large intestinal epithelial surface, but they bind to a subset of CD45+ leukocytes ( Figure 2).

Conclusions
Direct intestinal infection by seasonal infl uenza viruses seems an unlikely explanation for the frequent fecal detection of viral RNA in the patients reported here. No clinical correlation was shown for RNA positivity (but was shown with lymphopenia and positive virus isolation in NPA, indicating higher virus load), and culture positivity is rare (4,5,10,11   Three outliers were omitted from the fi gure for better illustration. Fecal viral RNA concentration was determined by using quantitative real-time reverse transcription PCR specifi c for the viral matrix gene and was expressed as log 10 RNA copies/g of stool. The lower detection limit of the assay was 3.7 log 10 RNA copies/g of stool. not found to express on normal intestinal epithelial cells (12). These fi ndings agree with reports which showed that intestinal cells and tissues do not support effi cient replication of seasonal viruses (12,13), thus their low potential to cause direct intestinal infection. Alternatively, swallowing of virus-containing nasopharyngeal secretions (although it seems inadequate to explain the higher rate of detecting fecal viral RNA than RSV or PIV) and hematogenous dissemination to organs through infected lymphocytes or macrophages in severe infl uenza cases with high virus load (spillover) are possible explanations for fecal viral RNA detection (2,14). Our fi ndings on virus receptor distribution and in vitro virus binding to intestinal lamina propria leukocytes lends support to the latter hypothesis. Notably, viral RNA positivity in nonpulmonary tissues infi ltrating mononuclear cells without detectable viral particles or antigens or tissue damage has been reported (15). Our study does not reject the possibility of seasonal infl uenza viruses causing occasional, disseminated infection in profoundly immunosuppressed persons because receptor affi nity is not absolute (2). Conversely, highly pathogenic avian infl uenza (H5N1) and pandemic (H1N1) 2009 viruses have the ability to bind to avian-like infl uenza virus receptors on colonic epithelium and to replicate effi ciently in intestinal cells and tissues (12). Their enhanced potential to cause direct intestinal infections and fecal-oral transmission deserve further investigation.