Figure 1 presents a flow chart showing the strategy of virus screening and case inclusion or exclusion. In this study, a total of 2, 262 respiratory tract specimens were collected from pediatric patients with ARTI who were admitted to the Department of Respiratory Medicine from June to December 2018, of which 105 specimens were positive for HAdV, as determined by DFA and/or RPP assays. After removing duplicates, a total of 1, 840 cases were included for further analysis, including 266 patients admitted to the hospital in June, 246 in July, 255 in August, 274 in September, 247 in October, 262 in November, and 290 in December (Fig. 2A). Among these, 95 were HAdV-positive (5.2%, 95/1840), of which only 81 individual cases, including 56 males (69.1%) and 25 females (30.9%), were genotyped for HAdV by PCR combined with phylogenetic analysis for viral homology of 301-bp fragments of the hexon gene, including seven cases positive for HAdV1 (8.6%, 7/81), 30 for HAdV3 (37.0%, 30/81), two for HAdV6 (2.5%, 2/81), and 42 for HAdV7 (51.9%, 42/81); 14 individual cases were not subjected to phylogenetic analysis due to insufficient PCR products for DNA sequencing.
Figure 1. The flow chart of the critical of cases inclusion or exclusion and distinguishing nosocomial HAdV infection from community infection.
Figure 2. Monthly distributions of HAdV positive cases during June to December, 2018 in the study. A Numbers tested or Numbers positive for HAdV; B Numbers of case positive for different HAdV types.
Using three pairs of universal PCR primers that target the variable regions of the three major capsid genes, we obtained 79 sequences of the penton gene, 75 sequences of the hexon gene, and 66 sequences of fiber gene (Supplementary Figure S1) among those 81 individual cases with 301-bp hexon gene fragments. There were two cases (1W799 and D1476) with three capsid gene sequences belonging to different HAdV types, which might have been due to infection by recombinants or co-infection by different HAdV types, one case (D1909) without any sequence corresponding to those three capsid genes, and 78 cases with sequences in accordance with the genotyping results of the 301-bp hexon gene fragments. Therefore, we selected the genotyping results of the 301-bp fragments of the hexon gene for further analysis.
In June, there were four cases (1.5%, 4/266) of HAdV infection, including one with HAdV1, two with HAdV3, and one with HAdV7, whereas there was only one case (0.4%, 1/246) of HAdV3 infection in July. However, cases of HAdV infection increased rapidly in August with 12 cases (4.7%, 12/255), including five HAdV3, one HAdV6, and six HAdV7, as well as in September with 41 cases (15.0%, 41/274), including three HAdV1, six HAdV3, one HAdV6, 23 HAdV7, and eight unknown. Thereafter, HAdV infection cases decreased gradually, with 17 cases (6.9%, 17/247) in October, consisting of one HAdV1, six HAdV3, seven HAdV7, and three unknown, 10 cases (3.8%, 10/262) in November, including one HAdV1, five HAdV3, two HAdV7, and two unknown, and 10 cases (3.4%, 10/290) in December, comprising one HAdV1, five HAdV3, three HAdV7, and one unknown (Fig. 2B).
Based on phylogenetic trees of the 301-bp fragment of the hexon gene, sequences of seven cases were typed as HAdV1 in three clusters, 30 as HAdV3 in five clusters, two as HAdV6 in two clusters, and 42 as HAdV7 in eight clusters (Fig. 3). According to clinical information and data of phylogenetic analysis, those 95 cases, including 81 with sequences in phylogenetic trees and 14 without sequences, were grouped step-by-step as shown in Fig. 1. There were 46 cases of patients admitted to the Department of Respiratory Medicine for the first time and confirmed to be HAdV-positive within 48 h after admission, including 40 in phylogenetic trees (in blue, Fig. 3; 1W799, D1476, and D1909 were included) and six without sequences, whereas there were 22 cases with specimens collected within 48 h after admission with more than 8 days isolation from the previous discharging, including 17 in phylogenetic trees (in green, Fig. 3) and five without sequences. All of these 68 (71.6%, 68/95) cases were not considered nosocomial infections.
Figure 3. Phylogenetic trees of 301 bp fragments of hexon gene constructed using MEGA X software to identify the genotypes of HAdV-positive specimens. Sequences from the study were named as "No. of specimens–Date of admission–Date of discharging–No. of ward and bed–Typing of HAdV". Sequences from GenBank were labelled with their GenBank number- typing of HAdV, which were in different clusters (Cluster 1–8) in different HAdV types. Blue: sequences from cases admitted to the Department of Respiratory Medicine for the first time and were confirmed to be HAdV positive within 48 h after admission. Green: sequences from cases with specimens collected in 48 h after the admission with over 8 days isolation from the previous admission. Orange: sequences from cases who were admitted over one time and confirmed with sequences in different sero- or geno-type or in different clusters of one type from their roommates positive for HAdV during their previous admission. Yellow: sequences from cases confirmed to be HAdV after 48 h from admission shared no homology sequence with their roommates. Red: sequences from case owning a roommate positive for HAdV, but without HAdV sequence. Purple: sequences from cases with HAdV sequences shared high homology with that of roommates in the previous or this admission.
Among cases of patients admitted to the Department of Respiratory Medicine more than once, there were nine cases (in orange, Fig. 3), including five cases with 7 days, two cases with 6 days, one case with 4 days, and one case with 3 days of isolation from the previous discharging. These were confirmed to be associated with sequences of different genotypes or different clusters of one type, as compared to those of their HAdV-positive roommates during the previous admissions of these nine patients. Therefore, these nine (9.5%, 9/95) patients might have been infected by the community. Among cases confirmed as HAdV 48 h after admission, there were eight cases (in yellow, Fig. 3) with no homologous sequences shared with those of their roommates, which implied that these patients might have been infected by visitors or attending parents. However, there were three cases from other wards with sequences sharing high homology with those of the eight cases. For these eight patients, most (5/8) might have been infected during Aug 15 to Sep 12.
There were 10 remaining cases, for which nosocomial infections could not be excluded. Among them, there were three groups. In group one, there were three cases without sequences that might have indicated infection by roommates positive for HAdV. In group two, there was one case (in red, Fig. 3) with a roommate positive for HAdV, but without an HAdV sequence. In group three, there were six cases (in purple, Fig. 3) with HAdV sequences that shared high homology with those of roommates during their previous or current admission, which was supported by the high homology of three capsid gene sequences shared with those of their roommates (Supplementary Figure S1). For these 10 cases, most (6/10) might have also been infected during Aug 15 to Sep 12 (Table 1).
Groups No. The previous admission The admission Date confirmed as HAdV positive Sero- or Geno-type of HAdV Cluster Date of admission Date of discharge No. of bed Date of admission Date of discharge No. of bed 1 D1334 – – – 8/18 9/7 66, 23, 21 9/3 Unknown Unknown D1834 – – – 11/27 12/5 46 12/2 Unknown Unknown D1380 8/26 9/8 42 9/11 10/22 59 9/11 Unknown Unknown 2 D1873 – – – 11/30 12/18 44 12/10 HAdV7 1 3 1w300 – – – 9/26 10/19 69, 9 10/11 HAdV7 1 D1376 – – – 9/4 9/19 48 9/11 HAdV7 1 99533 9/3 9/7 30, 8 9/11 9/27 63 9/11 HAdV3 1 D1399 8/28 9/7 34 9/13 9/22 69 9/13 HAdV7 1 D1419 9/1 9/12 31 9/16 10/9 8 9/16 HAdV7 1 D1656 10/13 10/19 31 10/26 11/7 70 10/26 HAdV7 1 Groups Possible date of infection Nosocomial infection was considered Possible sources of infection No. Sero- or Geno-type of HAdV Cluster Date of admission Date of discharge No. of bed 1 8/18–9/3 Yes 99180 HAdV-7 1 8/15 9/7 61 98638 HAdV-6 1 8/10 8/23 68 11/27–/12/2 Yes D1806 Unknown Unknown 11/27 12/11 50 8/26–/9/8 Yes D1302 HAdV-7 5 8/16 9/4 44 2 11/30–12/10 Yes D1806 Unknown Unknown 11/27 12/11 50, 40 3 9/26–10/10 Yes 99943 HAdV-7 1 9/26 10/8 70 9/4–9/11 Yes D1341 HAdV-7 1 9/1 9/8 49 9/3–9/7 Yes 99416 HAdV-3 1 9/5 9/8 32, 8 8/28–9/6 Yes 99058 HAdV-7 1 8/27 9/8 34, 9 9/1–9/12 Yes 99547 HAdV-7 1 9/11 9/10 33, 6 10/13–10/19 Yes 1w671 HAdV-7 1 10/11 10/24 34
Table 1. Information of the ten cases for whom nosocomial infection were considered.