a total of 909 remnant fecal samples were collected from 419 patients ; 473 samples from 220 patients were collected during january  december 2008 , and 436 samples from 199 patients were collected during january 2010june 2011 ( technical appendix figure 1 ) . all samples were de - identified , so that only a patient identification code , sample identification code , and date were known . jude children s research hospital institutional review board approved this study with a waiver of consent . we extracted viral rna from homogenized fecal samples by using the magmax-96 viral rna isolation kit ( applied biosystems life technologies , carlsbad , ca , usa ) . we screened samples from 2008 by using a singleplex real - time reverse transcription pcr ( rrt - pcr ) ( 3 ) , an in - house multiplex pcr to identify canonical ( hastv18 ) and noncanonical ( mlb1 and va2 ) genotypes ( 4 ) , and endpoint pcrs by using primers targeting open reading frame ( orf ) 1(5 ) and orf2 ( 6,7 ) . the singeplex method detected hastv in 23/72 samples , compared with 61/67 ( 5 samples were not tested ) by the multiplex method . we screened samples from 20102011 solely by the multiplex method ; 14 were positive for hastv . overall , we detected hastv in 86 ( 9.5% ) of 909 samples from 60 ( 14.3% ) of 419 patients ( table ; technical appendix table ) , giving a detection rate comparable to those reported for other immunocompromised populations ( 8,9 ) . * hastv , human astrovirus ; i d , identification ; nd , not detected ; und , undetermined ; rrt - pcr , real - time reverse transcription pcr . to place these findings in the context of those for other enteric virus infections , we also used rrt - pcr methods to determine the prevalence of norovirus and sapovirus in the fecal samples from 2008 ( 10,11 ) . due to sample quantity limitations we detected hastv in the highest proportion of patients and samples ( 22% [ 49/220 ] and 15% [ 72/473 ] , respectively ) , followed by norovirus ( 11% [ 39/220 ] and 12% [ 58/473 ] ) and sapovirus ( 12% [ 25/211 ] and 6.6% [ 29/442 ] ) ( figure 1 ) . together , these data demonstrate that hastvs are a major contributor to the enteric virus infections in this patient population . furthermore , co - infection occurred in only 16% of hastv - positive patients ( table ; technical appendix table ) , much lower than the 33%65% of patients reported in other studies ( 2 ) . enteric virus infections identified from remnant fecal samples from pediatric patients with cancer , memphis , tennessee , usa , 2008 . the percentage of samples and patients testing positive for human astrovirus was higher than the percentages testing positive for either norovirus or sapovirus . * due to limited sample availability , 31 samples could not be tested for sapovirus . a median of 40 ( range 1854 ) samples were collected each month in 2008 ; cases peaked in spring and decreased in summer and early fall ( technical appendix figure 2 ) . we further investigated temporal trends in hastv infections by using longitudinal samples from 34 patients ( 212 samples / patient ) collected over a median of 81 ( range 3328 ) days . of the 34 patients , 12 were previously hastv negative , indicating that more than one third of patients had newly acquired infections ( technical appendix table ) . of the 12 patients with > 1 positive specimen , 6 experienced prolonged hastv shedding ( defined by positive samples collected > 2 weeks apart [ range 17183 days ] ) ( technical appendix table ) . subsequent fecal samples for 2 patients ( sj35 and sj209 ) were negative for hastv , suggesting that the virus had cleared , whereas the other 4 patients ( sj22 , sj175 , sj245 , and sj275 ) had detectable hastv in their final fecal samples . despite being co - infected with hastv and norovirus , patient sj275 persistently shed only hastv , highlighting the need to further explore enteric virus co - infections and the potential for virus interference . we genotyped 50 of the 86 hastv - positive samples by using the aforementioned endpoint pcr methods , with patient - specific primers and the 3 race system ( invitrogen life technologies , carlsbad , ca , usa ) for rapid amplification of cdna ends to obtain partial open reading frame ( orf ) 1b ( rna - dependent rna polymerase ) and orf2 ( capsid protein ) sequences . we were unable to genotype the remaining positive samples because of inadequate sample quality or quantity . we used 50 sequences , collectively representing 38 unique infections , in the phylogenetic analysis ( technical appendix figure 3 ) and blast ( https://blast.ncbi.nlm.nih.gov ) searches for samples with only shorter sequence reads available . tree topology of orf1b and orf2 sequences indicated that none of the samples contained recombinant viruses or was co - infected with different genotypes . overall , hastv1 was the most prevalent genotype identified ( n = 19 , 50% ) , followed by the noncanonical genotypes va2 ( n = 8 , 21% ) and mlb1 ( n = 5 , 13% ) ( figure 2 ) . fecal samples from 4 ( 11% ) patients were positive for hastv2 ; 1 each was positive for hastv5 and hastv8 . of note , 4 patients were sequentially infected with > 1 genotype , and 3 patients from 2008 had serial infections with canonical and noncanonical genotypes ( table ; technical appendix table ) . co - circulating human astrovirus ( hastv ) strains in pediatric patients with cancer in memphis , tennessee , usa . six different hastv genotypes were identified among the hastv - positive fecal samples collected from pediatric patients in 2008 or in 20102011 ; more than one third of the viruses were noncanonical va and mlb genotypes . in a previous study of immunocompromised children hospitalized with diarrhea , the prevalence of hastv infection was 5% ( 9 ) . hastv detection in hiv - infected persons of all ages with and without gastroenteritis has been reported to be as high as 12% ( 8) . thus , the 14% detection rate we observed in pediatric oncology patients is consistent with the rate in previous reports . some patients showed prolonged virus shedding , which has been reported in immunocompetent and immunocompromised children , in some cases for as long as 3 months ( 12,13 ) . however , 4 of the patients in our study shed virus beyond 3 months , including 1 who shed for 6 months , highlighting the ability of hastv infections to persist within this population . in addition , 3 of the 4 patients with prolonged shedding were infected with the noncanonical genotypes mlb1 or va2 . although genotypic differences in virus load and long - term shedding have been reported for canonical viruses ( hastv18 ) ( 12 ) , we are not aware of such studies with noncanonical viruses . a larger , prospectively followed , longitudinal cohort would be required to investigate these differences . our identification of 4 patients with sequential hastv infections is notable , especially because all cases occurred during 1 year of observation . one previous report also described a child who was first infected with hastv3 and then , 9 months later , with hastv1 ( 14 ) . although our study showed more than one third of the hastv infections detected in samples from 2008 were newly acquired , we do not know the precise circumstances of these infections  whether they were associated with healthcare settings , or if they occurred at particular times during the patients treatment . the trend of hastv infections in our study appears to mirror that in reports from egypt , brazil , and the eastern united states , which also observed a peak of infections in late spring and early summer ( 13 ) . in summary , we used multiple molecular methods to identify hastv infections in immunocompromised pediatric oncology patients , enabling us to examine a broad representation of the infections experienced by this population and to identify 6 co - circulating viruses . we showed that the singleplex rrt - pcr method ( 3 ) was unable to capture infections caused by noncanonical viruses and is , therefore , limited in its utility for accurate surveillance and diagnosis . although our multiplex method was able to identify all canonical and 2 noncanonical viruses ( 4 ) , it is possible that we missed infections with other strains . given the limited knowledge about hastv genotypes and clinical disease , particularly in immunocompromised persons , additional studies will be crucial to help develop better diagnostics to capture all known hastv genotypes . characteristics of human astrovirus ( hastv)positive samples , pipeline for hastv detection , temporal distribution of hastv and phylogenetic trees of open reading frame 1b and 2 sequences .
human astroviruses are a major cause of pediatric gastroenteritis , especially in immunocompromised children . 
 we conducted a retrospective study to demonstrate that diverse astrovirus genotypes can co - circulate in pediatric oncology patients . 
 a subset of cases is associated with long - term virus shedding ( range 17183 days ) .