Hepatitis B virus genotype E infection among Egyptian health care workers

Study population

A cross-sectional study was conducted between June 2014 and April 2015 among the workers of governmental (n = 461) and non-governmental (n = 103) hospitals in Tanta City, Egypt. These included both tertiary (n = 257), secondary (n = 191) and primary care hospitals (n = 116). All currently employed staff were invited to the study during an eight-week period. All those reporting for duty during the study period were eligible for enrollment. Medical and nursing students on clinical placements were not considered as hospital staff, and hence, were ineligible for recruitment.

Study recruitment

The recruitment of HCWs was based on the study enrollment acceptance, without any random selection. Overall, 564 HCWs completed both the study questionnaire and provided a blood sample for testing. A single study-trained researcher collected data from all the study participants. Data on personal demographics and risk factors of HBV infection were collected.

Blood sampling

For each study participant, 5 mL of venous blood was collected. Each sample was centrifuged within six hours of the collection into two serum aliquots for storage at -21°C and further testing took place later.

Anti-viral serology

All Health Care Workers were tested for the presence of HBsAg and anti-HBc, by third generation enzyme-linked immunosorbent assay (ELISA). Anti-HBc positive samples were re-tested and only samples testing positive on the two occasions were considered positive.

Detection of Hepatitis B virus DNA by nested PCR

HBsAg and/or anti-HBc positive samples (n = 140), tested for HBVDNA using nested PCR techniques, as described previously.^[11] Briefly, DNA was extracted from the patients’ sera using QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany). HBV DNA was amplified using two different primer pairs (First set: F 5’- GTCTGCGGCGTTTTATC- 3’; and R 5’-ACAGTGGGGGAAAGC- 3’; second set: F 5’ - TGCCCGTTTGTCCTCTA- 3’ and R 5’ -AGAAACGGRCTGAGGC- 3’). Samples from all positive cases were retested to confirm the positive results. The first round of PCR was performed with an outer primer set for 35 cycles (94 °C for 40 s, 55 °C for 40 s, and 72 °C for 40 s). The second round was performed with an inner primer set for 25 cycles (94 °C for 40 s, 57 °C for 40 s, and 72 °C for 40 s), followed by the extension reaction. Nested PCR products were subjected to electrophoresis on a 3% agarose gel stained with ethidium bromide, and DNA was observed under ultraviolet light. The detection limit of our nested PCR was 10 copies/mL.

Hepatitis B virus genotyping

A total of 14 HBVDNA-positive serum samples by nested PCR underwent HBV genotyping. The genotyping procedure and sequences of PCR primers were conducted as described before.^[12,13]

All procedures performed in the study were approved by Al-Zagazig University Ethics Committee, and in concordance with the 1964 Helsinki Declaration and its later amendments. Informed consent was obtained from all individual participants included in the study.

Statistical analysis

SPSS version 17 was used for analysis. Differences in frequency between groups were compared with the chisquare test or the Fisher exact test. A P value < 0.05 was considered significant.

Demographic data

Five hundred and sixty-four (564) HCWs were enrolled, relating to both governmental (n = 461) and nongovernmental hospitals (n = 103) in Tanta City (Table 1). While 258 (45.74%) of the included workers provided direct health care to patients, 306 (54.25%) were non-health care providers (Table 1). Among the included HCWs, 319 (56.56%) were males and 245 (43.44%) were females, with a mean age of 33.0 ± 9.8 years (range 16–64). The mean duration of health care work was 9.3 ± 6.7 years (range 1–30). 9 (1.6%), 13 (2.3%), 15 (2.7%) and 17 (3%) HCWs had a history of HCV infection, parenteral antischistosomal therapy (PAT), history of type II diabetes mellitus and prior surgery, respectively. Forty-five (8%) HCWs had a history of infantile HB vaccination. None of the included HCWs had a history of adult HB vaccination, blood transfusion or hemodialysis (Table 1).

HBsAg serology

The frequency of HBsAg among the included HCWs was 1.4% (8/564) (Table 2). Of them 62.5% (5/8) worked at governmental hospitals and 37.5% (3/8) worked at nongovernmental hospitals. Of the eight HBsAg positive subjects, two worked at security, two worked as ambulance drivers, one worked at administration department and three were nurses (Table 2). The frequency of anti-HBc positivity among HBsAg positive HCWs was 75% (6/8), all of them have positive viremia. Thus, two (25%) of the eight subjects with positive for HBsAg were negative for anti-HBc.

Anti-HBc serology

The frequency of anti-HBc among the included HCWs was 24.5% (138/564). Of those, 51.8% (73/138) worked in governmental hospitals, while 47.5% (66/138) worked in non-governmental hospitals. Anti-HBc positive rate in health care providers and none health care providers were 23.64% (61/258) and 25.16% (78/306), respectively. One (33.3%), three (18.75%), and 57 (23.84%) of the included physicians, laboratory technicians, and nurses were seropositive for anti-HBc respectively. The distribution of anti-HBc sero-positivity among ward workers, officers, security workers, drivers, and cooks were 21.9% (34/155), 25% (17/68), 32.5% (13/40), 32.35% (11/34), and 22.2% (2/9), respectively (Table 2). Older HCWs and those with a longer duration of health care work had significantly higher rates of anti-HBc sero-positivity (Table 3).

HB viremia among HB infected HCWs

Among 140 HCWs positive for HBsAg and/or anti-HBc, HBVDNA was detected in 14 (10%) by nested PCR. 8 (57.14%) HCWs were working in governmental hospitals while 6 (42.85 %) were in non-governmental hospitals (Table 2). 6 (2.51%) nurses and 8 (2.61%) non-health care providers were positive for HBVDNA (Table 2). While the frequency of positive viremia among HBsAg positive HCWs was 87.5% (7/8), it was 8.7% (12/138) among anti-HBc positive. Among the two patients with HBsAg positive but anti-HBc negative, HBVDNA tested positive.

HBV genotypes

Among 14 HBVDNA positive HCWs, HBV/E (n = 7), HBV/D (n = 3) and co-infection with E and D (n = 4) genotypes were detected. Genotype E was detected in governmental and non- governmental HCWs. The mean age of HCWs with HBV/D, HBV/E, and co-infected HBV/D and E genotypes were 28 ± 7.93, 34 ± 8.5 and 48.5 ± 8.26 years, respectively. In addition, the mean duration of health care work for those with HBV/E, HBV/D and co-infection with E and D were 6 ± 4.35, 8.7 ± 5.99 and 18.5 ± 7.85 years respectively.

HBV infection among HCWs with infantile HB vaccination

Forty-five (7.8%) HCWs were born after 1992, and thus received infantile HB vaccination. Their mean age was 19.35 ± 1.53 years; there were 21 (46.66%) males and 24 (53.33%) females. Although none was positive for HBsAg, 14 (31.11 %) were anti-HBc-positive and of them, 14.3% (2/14) were HBVDNA positive. Among them, both genotypes D (n = 1) and E (n = 1) were detected.