Distribution of patients according to age and size of urban centers
According to official data of 2011 census, urban centers were classified in large to medium centers (LMCs) (>30,000 inhabitants, range 44,000–1,203,000) and small centers (SCs) (<30,000 inhabitants, range 132–27,000). One hundred and five patients with acute HAV infection were evaluated in the study. Among them, 46 (43.8%) were from large to medium centers (LMCs) and 50 47.6%) from small centers (SCs). Nine subjects (8.6%), of unknown nationality, were from a refugee camp in Bulgaria. The age was known for 102 patients and their classification into three age categories (Children: 0–12; Teens: >12 to 19; Adults: >19) indicated that 48 (47.1%) were children, 13 (12.7%) teens and 41 (40.2%) adults.
Distribution of hepatitis A cases according to the size of urban centers revealed an opposite age distribution pattern in LMCs and SCs (Fig. 1). The percentages of children and adults were 33.3% and 53.3%, respectively, in patients from LMCs, while they were 54.0% and 32.0% in patients from SCs (p < 0.05, Fisher’s exact test). The percentage of teens was similar in the two groups.
Genotyping of HAV strains
Phylogenetic analysis of the 105 HAV isolates from Bulgaria and reference sequences with known genotype revealed two main groups corresponding to the IA (n = 78) and IB (n = 27) sub-genotypes (Fig. 2). No genotype II or III isolates were observed.
In the IA group two main statistically supported clusters can be observed (Fig. 2, labelled as “MAJOR cluster” and “minor cluster”). The major cluster included sequences (n = 62, 79.5% of IA isolates) showing a very high intra-cluster identity level (mean value 99.6%¸ range 98%–100%).
The minor cluster contained IA sequences (n = 16, 20.5% of IA isolates) characterized by a lower intra-cluster identity level (mean value 98.1%¸ range 93.7%–100%).
In the IB group, the 27 sequences showed a high identity level (mean value 99.1%¸ range 98%–100%). Among the IB isolates from 9 patients living in a refugee camp in Bulgaria, 2 small clusters (each containing 3 isolates) of identical sequences were observed. Although the sequences from refugees showed a tendency to cluster separately from the other IB isolates, bootstrap values were not significant: sequences from refugees showed high identity (1 to 4 nt differences) with several sequences from Bulgarian patients circulating in the same (2014) and in the previous years (2012 and 2013) suggesting that refugees got infected with local strains.
To include in the phylogenetic analysis also the proposed sub-genotype IC [17], a separate analysis limited to a shorter region (261 nt) had to be carried out; in fact the available reference IC sequences only partially overlapped with the region sequenced in the present study. IC isolates formed a separate cluster, intermediate between IA and IB; however, none of the Bulgarian isolates grouped with them (data not shown).
Nucleotide variations in strains from the IA major cluster
To better compare HAV isolates from the IA major cluster, one of them, the ISS49_DoB, was chosen as reference sequence. Based on this, the 62 isolates from the major cluster were classified into three groups as follows (see also Fig. 2): (1) “strain 1” group, including 22 (35.5%) sequences (21 sequences showing 100% identity with ISS49_DoB plus the reference sequence itself); (2) “strain 2” group, including 22 (35.5%) sequences presenting only 1 nucleotide (nt) difference with ISS49_DoB, with all isolates showing the same nt difference in the same position (nt 403, C → T, numeration of [GenBank:KF182323]); strain 2 is identical to a strain (reference sequence [GenBank:KF182323] in Fig. 2) previously reported to be responsible for both an outbreak in the Czech Republic in 2008 and a large European outbreak caused by mixed frozen berries in 2013 [15]; (3) “strains related to strain 1 and 2” group, including 18 (29%) sequences showing 1 to 4 nucleotide differences versus ISS49_DoB (individual nucleotide variations were scattered in different positions) (Fig. 2, see the box). Overall, strain 1 and strain 2 represent 44/62 (71%) isolates of the major cluster; 12 distinct variants could be recognised in the remaining 18/62 (29%) isolates of this cluster.
By linking epidemiological and sequence data, a small outbreak of 9 cases could be clearly identified in the major cluster. All cases were from Ihtiman village and occurred between 25 June and 8 August 2012, involving 6 children and 3 teenagers: the complete identity of the isolates confirms a local small outbreak caused by the strain 1 during a period of two months (Fig. 2, MAJOR cluster, labelled by the asterisk).
Distribution of HAV genotypes and strains in urban centers of different size
Distribution of HAV genotypes according to the size of the town/village of patients showed that the percentages of isolates belonging to IA major cluster, IA minor cluster and IB group were 42.2%, 31.1% and 26.7%, respectively, in patients from LMCs and 86.0%, 2.0% and 12.0% in patients from SCs (Fig. 3a). Most isolates from the IA major cluster (43/62, 69.4%) were identified in patients from SCs. In contrast, all but one strains from the IA minor cluster were observed in patients from LMCs, suggesting that different transmission routes, linked to urbanization, operate in these cases. Among the IB sub-genotype, isolates were identified in both LMCs and SCs, however the frequency was higher in patients from LMCs (26.7% in LMCs vs 12% in SCs) (Fig. 3a). Analysis of the 62 isolates from the IA major cluster revealed a different frequency of strain 1 and strain 2 in LMCs and SCs (Fig. 3b): strain 1 largely prevailed in SCs (5.3% in LMCs vs. 48.8% in SCs), strain 2 prevailed in LMCs (63.1% in LMCs vs. 23.3% in SCs) (p < 0.001, Fisher’s exact test); in contrast, the strains related to strain 1 and 2 showed similar frequency (31.6% vs. 27.9%).
Analysis of the geographical localization of the strains in Bulgaria map showed that strain 1 was preferentially detected in a restricted area south-east of Sofia, strain 2 and strains related to strain 1 and 2 showed a more scattered distribution, whereas most strains from the IA minor cluster and IB strains were observed in large towns such as Sofia, Pernik, Shumen, Gabrovo and Varna (Fig. 4).