1. The relative importance of respiratory viruses in lower respiratory tract infections in primary care. [P1581] F. Coenjaerts 1, C. Lammens 2, M. Viveen 1, L. Tan 1, K. Loens 2, H. Goossens 2, P. Zuithoff 3, M. Ieven 2, A. van Loon 1, and E. Claas 4 on behalf of the GRACE study team 1 Univ. Med. Centre Utrecht – Dept. of Med. Microbiology, The Netherlands; 2 University of Antwerp, Belgium; 3 UMC Utrecht – Julius Center; 4 UMC Leiden, the Neth. Objectives: The microbial etiology and especially the role of viruses in adult lower respiratory tract infections (LRTI) in the community is not well known. We therefore investigated the viral etiology in LRTI at the GP’s office in the European GRACE primary care network (PCN) using sensitive real-time PCR (RT-PCR). Patients: From October 2007 through May 2010, a total of 3,018 adult patients (V1) with LRTI in the community were enrolled in a prospective study in 14 PCNs in 12 European countries (Figure 1). Samples: Nasopharyngeal flocked swabs (COPAN) were collected and analyzed for the presence of influenzavirus (INF) A/B, respiratory syncytial virus (RSV), parainfluenzavirus (PIV) 1-4, human rhinoviruses (HRV), human metapneumovirus (hMPV), Bocavirus (Boca), coronaviruses (COR) OC43, NL63, 229E, adenovirus (ADE), as well as the novel polyomaviruses KI and WU. V0 samples (1185) were taken from age-, sex-, and geographically-matched controls; V2 samples (2544) were collected from the same patients – 4 weeks after V1. Here, we present primary etiological data and report on the relative importance of quantitative measurements (Ct values), as well as the aetiology of double infections. Analysis: Specimens were transported to the central lab in Antwerp for nucleic acid extraction by the NucliSens EasyMAG (bioMerieux); isolates were analyzed by RT- PCR either directly (DNA viruses) or after cDNA synthesis by using a Multiscribe reverse transcriptase kit together with random hexamers (Applied Biosystems). Viral loads were determined by the number of amplification cycles needed for a positive TaqMan realtime PCR test (cycle treshold, CT). Samples were counted positive for CT<40. INTRODUCTION MATERIALS & METHODS CONCLUSIONS Presentation rates of respiratory pathogens in V1 samples significantly exceed those found in controls (V0) and follow up (V2) samples; this does not hold true for Boca-, WU and KI virus, which might question them as true respiratory pathogens. The availability of quantitative viral load data is unlikely to affect individual patient management. Double infections do not affect patient presentation rates; even in control patients follow up visits double infections do occur. Table II NMean Statistic Std. Error Adeno_V1 3835,741,05 Adeno_V2 4237,810,59 Adeno_V0 1737,410,28 BOCA_V1 1235,381,77 BOCA_V2 1138,071,08 BOCA_V0 1637,640,87 COR_V1 20528,090,46 COR_V2* 7031,850,80 COR_V0* 2032,421,35 COR229E_V1 4227,410,78 COR229E_V2 1529,501,57 COR229E_V0 530,292,35 CORNL63_V1 3526,930,86 CORNL63_V2 1329,511,99 CORNL63_V0 429,911,59 COROC43_V1 8825,73,64 COROC43_V2* 2229,741,15 COROC43_V0 427,413,24 hMPV_V1 12430,040,37 hMPV_V2 733,141,56 hMPV_V0 332,381,32 INF_V1 29729,530,35 INF_V2 1029,551,60 INF_V0 428,903,04 INFA_V1 13328,990,43 INFA_V2 528,172,90 INFA_V0 326,081,61 INFB_V1 9826,860,51 INFB_V2 331,032,70 INFB_V0 0 -- KI_V1 2736,460,60 KI_V2 2835,90,89 KI_V0 1535,941,41 PIV1_V1* 1429,981,156 PIV1_V2 337,48,66 PIV1_V0 232,875,87 PIV2_V1 1128,651,61 PIV2_V2 232,504,50 PIV2_V0 136,91- PIV3_V1* 2328,831,25 PIV3_V2 736,091,63 PIV3_V0 236,732,82 PIV4_V1 2230,420,86 PIV4_V2 0 -- PIV4_Vo 131,07- Rhino_V1 57230,180,21 Rhino_V2* 11131,460,50 Rhino_V0* 5432,130,53 RSV_V1 14329,1300,52 RSV_V2 1330,531,57 RSV_V0* 735,771,69 WU_V1 4437,810,53 WU_V2 5438,750,31 WU_V0 2038,960,35 Table I V0 % pos. V1 % pos. V2 % pos. RSV0,5*3,60,4* INF0,2*7,40,3* INFA0,1*3,70,1* INFB0*2,90* COR1,5*5,41,4* 229E0,3*1,30,4* NL630,2*1,00,3* OC430,4*2,60,6* WU0,2 0,1 KI0,2 PIV10,1*0,40* PIV20,1*0,30* PIV30,2*0,60,1* PIV40,1*0,60* HRV3,6*15,62,7* hMPV0,3*3,70,2* ADE0,2*0,40,1* BOCA0,030,10,04 Table III V2COR 229E COR NL63 COR OC43 WUKIHRVADE V1 INFA0,063 KI0,017 PIV30,030,004 PIV40,093 HRV0,07 hMPV0,090,08 ADE0,009 RESULTS This project is supported through Priority 1 (Life Sciences, Genomics and Biotechnology for Health) of European Union's FP6, Contract number: LSHM-CT-2005-518226 Correspondence: f.e.j.coenjaerts@umcutrecht.nl www.grace-lrti.org TABLE I.Most viruses were present at significantly higher rates in V1 samples when compared to V0 and V2. Boca, WU and KI presentation rates however, did not differ between V0, V1 and V2. This finding, added to the extremely low number of samples positive for these viruses raises doubt to their qualification as respiratory pathogens. TABLE II.DNA viruses presented with significantly lower viral loads (average CT 37) when compared to RNA viruses (average CT 30; CT 27 for INF B and the various COR’s). Significant differences between patients, versus controls and follow up samples, were detected for RSV, COR (as a group), COR OC43, PIV1, PIV3 and HRV. TABLE III.PIV – in particular PIV3 and PIV4 - positive patients were most prone to a positive follow up. For PIV3, significant associations were detected with Cor 229E (P value = 0.03) and Cor OC43 (P = 0.004) as secundary pathogens. Also INF A, PIV4, HRV and hMPV are frequently followed by secundary viral infections. All other combinations (empty cells in Table III, as well as non-depicted combinations) did not shown any association. KI and ADE are significantly the most persistent (found in the same patient within a 4-week interval) viruses (P = 0.017 and 0.009, resp.). Double infections (not shown).Despite the fact that DNA viruses are detected substantially in respiratory specimen, their overall impact is overestimated since these viruses are largely present in V0 samples, V1-double infections, or V2 samples. WU (26.7%) and KI (21.8%) were most frequently involved in double infections. Of all positive V1 patients, 6.4% was double-infected, in V0 and V2 double infections reached 7.3 and 6.5%, resp. * Differing significantly from V1 Positive follow up (V2; columns) after specific V1 pathogens (rows); depicted P values indicating strong trends (P values <0.10) or significant associations (P values < 0.05). Figure 1. Primary Care Networks in GRACE.