Hepatitis B Antibodies

Blood cell counts were performed at the BNITM laboratories in Hamburg, Germany, or the BwKH in Hamburg, Germany, using a Cell Dyn 3200 device (Abbott, Chicago, Illinois, USA). Serology for hepatitis B [anti-HBc-(hepatitis B core-) antibodies, HBs-(hepatitis B surface-) antigen] was performed at the Institute for Hygiene and Environment in the City of Hamburg, Germany, using an Architect i1000 immunology analyser (Abbott). Blood analyses were based on standardized procedures in quality-controlled diagnostic laboratories.
Stool samples were either formalin-fixed [6 (link)] to look for protozoa and helminth eggs using microscopy at the BNITM in Hamburg, Germany, or the samples were used for DNA extraction without any additives. The PCR protocols used comprised both in-house and commercial approaches. The in-house approaches covered enteroinvasive bacteria (Salmonella spp., EIEC (enteroinvasive Escherichia (E.) coli)/Shigella spp., Campylobacter (C.) jejuni, and Yersinia spp.) in one single-tube multiplex real-time assay [1 (link), 2 (link)] and enteropathogenic protozoa (Entamoeba (E.) histolytica, Giardia (G.) duodenalis, Cryptosporidium (C.) parvum and Cyclospora (C.) cayetanensis in another single-tube multiplex real-time assay [3 (link)–5 (link), 7 (link), 8 (link)]. Additionally, PCR was used to detect soil-associated nematodes (Ascaris (A.) lumbricoides, Ancylostoma spp., Necator (N.) americanus, Strongyloides (S.) stercoralis) in a single-tube multiplex real-time assay [4 , 9 (link)] and African Schistosoma spp. (S. mansoni, S. intercalatum, and S. haematobium without discrimination on species level) in a final simplex real-time assay [10 (link), 11 (link)]. All in-house PCR procedures were performed after nucleic acid extraction using the QIAamp Stool DNA Mini Kit (Qiagen, Hilden, Germany) as described by the manufacturer and others [12 (link)]. Applied commercial PCRs comprised the RidaGene (R-Biopharm, Darmstadt, Germany) PCR kits “EAEC,”, “EHEC-EPEC”, and “‘ETEC-EIEC” targeting enteroaggregative E. coli (EAEC), enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxic E. coli (ETEC), and Shigella spp./EIEC and were performed as described by the manufacturer.
If available, incidence data of the notifiable infectious diseases from the German population from the year 2015 were used for comparison. Such data are published in the yearly report by the Robert Koch Institute in the German National Reference Centre for Infectious Diseases. For the parameters assessed in the study, the respective reference data were available for enteroinvasive bacteria and EHEC, the enteropathogenic protozoa G. duodenalis and Cryptosporidium spp. as well as for the hepatitis B virus [13 ].
If refugees refused sample acquisition, their decision was accepted. Accordingly, not all patients submitted samples.

Liver biochemical parameters were determined by a biochemistry analyzer (7600 Series; Hitachi, Tokyo, Japan). Platelet was measured by Sysmex XN-2000 (Kobe, Japan). Serum HBsAg, anti-HBs, hepatitis B e antigen (HBeAg), hepatitis B e antibody (anti-HBe), and hepatitis B core antibody (anti-HBc) were detected using an enzyme-linked immunosorbent assay kit (ARCHITECT i2000 SR; Abbott Architect, USA). Serum HBsAg were retested (Roche Cobas e602; Roche, Switzerland) when it exceeded the upper linearity limit (250 IU/mL). HBV DNA was quantified by using a real-time PCR assay (DAAN Diagnostics, Guangzhou, China). Detection limits of HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, and HBV DNA were 0.05 IU/mL, 10 IU/L, 1 S/CO, 1 S/CO, 1 S/CO, 500 IU/mL, respectively. The upper limit of normal (ULN) of ALT has been defined as 40 U/L for women and 50 U/L for men. The ULN of bilirubin has been defined as 21 μmol/L for women and 26 μmol/L for men. The normal range of albumin was 40-55 g/L. In addition, hepatitis B core-related antigen (HBcrAg) was not detected due to the lack of serum samples.

An automatic blood cell analyzer (Coulter LH 750, Beckman, Fullerton, Calif, USA) was used to detect complete blood counts. Serum HBsAg, hepatitis B core antibody (anti-HBc), and HBeAg levels were assessed using the Abbott ARCHITECT® i2000SR platform found on a chemiluminescent microparticle immunoassay. The detection range of HBsAg was 0.05-250.0 IU/mL, and sera with HBsAg levels above 250.0 IU/mL were subsequently serially diluted at 1 : 500. A real-time polymerase chain reaction assay (DAAN Diagnostics, Guangzhou, China) was used to measure baseline serum HBV DNA with a 500 IU/mL lower detection limit. The last test of HBV DNA for distinguishing LLV and CVR was measured by Abbott m2000 RealTime assays (Abbott, Des Plaines, Ill, USA) with a lower detection limit of 10 IU/mL. Alanine aminotransferase, AST, TB, ALB, GGT, and creatinine levels were tested by an automatic biochemical analyzer (Hitachi 7600P, Hitachi, Japan).

Clinical information was collected from the electronic medical records system. In addition to general information (sex and age), peripheral blood parameters (neutrophil-to-lymphocyte ratio (NLR) and platelets), liver function indexes (albumin (ALB), γ-glutamyl transpeptidase (GGT), total bilirubin (TBIL), alanine aminotransferase (ALT)), and tumor biomarkers (α-fetoprotein (AFP), cancer antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA)) were retrospectively collected. HBV-related factors including HBsAg, hepatitis B virus e antigen (HBeAg), hepatitis B virus e antibody (HBeAb) and hepatitis B core antibody (HBcAb) were tested in all patients before surgery. The presence of MVI was obtained from the pathological reports after surgery. MVI was defined as tumor emboli, identified only by microscopy, in the vascular lumen lined with endothelial cells.