Bloodborne infections in dental practice: prevalence of markers and phylogenetic analysis of circulating strains

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Introduction

Human immunodeficiency virus (HIV) and hepatitis B (HBV) and C (HCV) viruses remain among the most dangerous blood-borne infections, posing a serious threat to global health. These pathogens can cause chronic diseases leading to serious complications, including liver cirrhosis, cancer and immunodeficiency^1–3. Their prevalence varies across different regions of the world, but the commonality of transmission routes leads to similar epidemiological patterns, making them particularly challenging for the medical community to combat⁴ [1]. The main routes of transmission of HIV, HBV and HCV are contact with infected blood and other biological fluids. Key risk factors include the use of non-sterile medical and cosmetic instruments, injection drug use, transfusion of non-tested blood, and unprotected sexual contact. Thus, these infections pose a particular risk to vulnerable populations: injection drug users; individuals requiring frequent medical interventions; and healthcare workers who regularly come into contact with biological fluids.

Dentists belong to a group at increased occupational risk of infection with bloodborne infections due to frequent contact with the blood and saliva of patients. The likelihood of infection depends on the prevalence of HIV infection and viral hepatitis among the population, as well as on compliance with infection control measures in clinics [2–4]. In addition to dentists, all medical personnel involved in the treatment of patients, including assistants, nurses, dental technicians, and orderlies, are at risk of blood-borne infections. Furthermore, the possibility of gross violations of sanitary and hygienic standards and anti-epidemic requirements during dental appointments cannot be ignored. In summary, it is particularly important to assess the epidemiological situation in specific regions and among dental patients, as this allows for the development of effective prevention strategies, including pre- and post-contact protection of medical personnel.

The aim of this study is to assess the prevalence of HIV, HBV and HCV infection markers among dental patients and to provide a molecular genetic characterization of the identified pathogens.

Materials and methods

The study material consisted of blood plasma samples obtained from 497 patients who sought dental care at medical institutions in St. Petersburg under the compulsory health insurance program. Women predominated in the study group (349/497 people, 70.2%) compared to men (148/497, 29.8%). The age of the subjects ranged from 18 to 94 years, with an average age of 57.6 years. To assess the possible relationship between the age of the subjects and the detected markers, stratification was performed according to the previously recommended method [5] into the following age groups: 18–29 years (n = 43), 30–39 years (n = 52), 40–49 years (n = 51), 50–59 years (n = 95), 60–69 years (n = 138), and 70+ years (n = 118).

The study was conducted with the informed consent of the patients. The research protocol was approved by the local Ethics Committee of the St. Petersburg Pasteur Institute (Approval No. 151 dated 21.09.2022).

The following serological markers were determined as part of the study: HBsAg, anti-HBs, anti-HBcore, anti-HBC, antigens and antibodies (Ag/Ab) to HIV. To ensure maximum reliability of serological screening, an extended protocol was used in this study: each marker was determined in parallel in two replicates using two commercial test systems from different manufacturers: DS-IFA-HBsAg, DS-IFA-ANTI-HBsAg, DS-IFA-ANTI-HBc, IFA-ANTI-HCV, DS-IFA-HIV-AGAB-SCREEN (NPO Diagnostic Systems, Russia) and Vectohep B-HBs-antigen, VectoHBsAg-antibodies, HepBest anti-HBc-IgG, Best anti-HCV, CombiBest HIV-1,2 Ag/Ab (JSC Vector-Best, Russia). Samples with discordant results in two test systems or repeated tests were subjected to independent retesting to obtain a final result. All positive and questionable results of the HBsAg and anti-HCV tests were verified using specialized confirmatory test systems HBsAg-confirmatory-IFA-BEST and Best anti-HCV-confirmatory test (JSC Vector-Best, Russia), respectively. The sensitivity of the test systems used in this study to detect HBsAg was 0.01 IU/mL. In cases where HIV Ag/Ab was detected, Western blotting was performed using the NEW BLOT BIORAD reagent kit (Bio-Rad Laboratories, USA) according to the manufacturer’s instructions.

Screening for molecular biological markers using the polymerase chain reaction (PCR) method was performed with preliminary DNA/RNA isolation using the commercial AmpliPrime Riboprep kit (Central Research Institute of Epidemiology, Russia). All samples were pre-concentrated by ultracentrifugation of blood plasma for 1 hour at 24,000g at a temperature of +4 °C. HBV DNA, HCV RNA, and HIV RNA was performed by PCR with hybridization-fluorescence detection in real time using the commercial AmpliSens HCV/HBV/HIV-FL kit (Central Research Institute of Epidemiology, Russia) according to the manufacturer’s instructions. For all samples with HBV DNA undetectable by the above method, additional testing was performed using a highly sensitive technique developed at the Pasteur Research Institute of Epidemiology and Microbiology in St. Petersburg [6]. This approach ensures the detection of HBV DNA in conditions of low viremia, including in HBsAg-negative hepatitis B.

The HCV NS5B, NS5A, NS3, Core were obtained using amplification on specific primers, with preliminary reverse transcription and subsequent sequencing, as shown earlier [7]. Nucleotide sequences of complete HBV genomes were obtained using nested PCR based on overlapping primer pairs, as shown earlier [8].

The amplification products were purified and evaluated for fragment length and concentration. The sequencing reaction was performed according to the instructions for the ABI PRISM BigDye Terminator v. 3.1 reagent kit (Applied Biosystems, USA), with forward and reverse specific primers in three replicates for each primer pair of each analyzed sample region in order to increase reliability. (Applied Biosystems, USA), with forward and reverse specific primers in three replicates for each primer pair of each analyzed sample region in order to increase data reliability, ensure sufficient read overlap, and level out possible artifacts, as specified in the Sanger sequencing and molecular testing quality control manual [9], as well as in a number of studies [10–12]. Nucleotide sequences were determined using an ABI Prism 3500 genetic analyzer (Applied Biosystems, USA) according to the manufacturer’s instructions. The electropherograms obtained from all repeats were visually inspected for quality, aligned, and a consensus sequence was constructed for each sample. The consensus sequences were used for subsequent phylogenetic and mutation analysis.

The obtained sequences were deposited in the international GenBank database, where they were assigned the corresponding numbers (PX091934–PX091945 (HCV), PX120108–PX120126 (HBV)).

Primary analysis of the consensus nucleotide sequences obtained was performed using the NCBI Blast program in comparison with the nucleotide sequences presented in the international GenBank database.

HCV genotyping was performed using phylogenetic analysis of Core and NS5B protein nucleotide sequences, as well as using the Hepatitis C Virus Phylogenetic Typing Tool v.2.11 software (https://www.genomedetective.com/app/typingtool/hcv/). To identify drug resistance mutations in the NS5B, NS5A and NS3 regions, we used the Genafor Geno2pheno HCV resistance software (https://hcv.geno2pheno.org/).

The obtained HBV sequences were genotyped based on phylogenetic analysis. Three online tools were used to identify clinically significant polymorphic variants [13]: the Stanford University HBVseq database program (https://hivdb.stanford.edu/HBV/HBVseq/development/HBVseq.html), HIV-GRADE HBV (https://www.hiv-grade.de/cms/grade/explanations/hbv-tool/), and Genafor Geno2pheno HBV (https://hbv.geno2pheno.org/).

Statistical data processing was performed using MS Excel and Prizm 5.0 (GraphPad Software Inc., USA). The Klopper–Pearson exact interval was used to assess statistical error. The results are presented with a 95% confidence interval (95% CI). To identify statistically significant differences between arrays comprising several groups, Pearson’s χ² criterion was applied to contingency tables. If a significant overall p < 0.05 was obtained, a post-hoc analysis was performed using Fisher’s exact test or the χ² test with Yates’s correction, depending on the size of the expected frequencies, to determine between which specific pairs of groups there were differences. A probability value of p < 0.05 was set as the threshold for significance.

Results

Prevalence and distribution of serological markers in the group

When assessing the overall prevalence of anti-HCV serological markers, they were detected in 3.8% (19/497, 95% CI 2.3–5.9%) of cases, while HIV Ag/Ab was detected in 1.2% (6/497, 95% CI 0.4–2.6%) of cases, with 2 samples confirmed by immunoblotting, accounting for 0.4% (2/497) of the total group and 33.33% (2/6) of the group with newly detected markers. Only 293 people did not have markers associated with HBV, accounting for 58.95% (293/497, 95% CI 54.5–63.3%). At the same time, HBsAg was detected in 2.4% (12/497, 95% CI 1.3–4.2%), anti-HBs in 32.0% (159/497, 95% CI 27.9–36.3%), and anti-HBcore in 25.6% (127/497, 95% CI 21.8–29.6%) of those examined. The prevalence of serological markers in different age groups is presented in Table 1.

 

Table 1. The prevalence of the analyzed serological markers across different age groups in the study population

Таблица 1. Распространенность анализируемых серологических маркеров среди обследованных разных возрастных групп

Age group Возрастная группа	Number of patients Число пациентов	Анти-HBs Anti-HBs		Анти-HBcore Anti-HBcore		HBsAg		Анти-ВГС Anti-HCV		Аг/Ат ВИЧ Ag/Ab HIV
	abs. абс.	abs. абс.	% (95% CI) % (95% ДИ)	abs. абс.	% (95% CI) % (95% ДИ)	abs. абс.	% (95% CI) % (95% ДИ)	abs. абс.	% (95% CI) % (95% ДИ)	abs. абс.	% (95% CI) % (95% ДИ)
18–29 years / лет	43	30	69.8 (53.9–82.8)	3	7.0 (1.5–19.1)	1	2.3 (0.1–12.3)	0	0.0 (0.0–8.2)	0	0.0 (0.0–8.2)
30–39 years / лет	52	19	36.5 (23.6–51.0)	10	19.2 (9.6–32.5)	0	0.0 (0.0–6.8)	1	1.9 (0.0–10.3)	1	1.9 (0.0–10.3)
40–49 years / лет	51	10	19.6 (9.8–33.1)	7	13.7 (5.7–26.3)	1	2.0 (0.0–10.4)	3	5.9 (1.2–16.2)	2	3.9 (0.5–13.5)
50–59 years / лет	95	23	24.2 (16.0–34.1)	24	25.3 (16.9–35.2)	2	2.1 (0.3–7.4)	5	5.3 (1.7–11.9)	1	1.1 (0.0–5.7)
60–69 years / лет	138	38	27.5 (20.3–35.8)	40	29.0 (21.6–37.3)	8	5.8 (2.5–11.1)	6	4.3 (1.6–9.2)	2	1.4 (0.2–5.1)
70 + years / лет	118	39	33.1 (24.7–42.3)	43	36.4 (27.8–45.8)	0	0.0 (0.0–3.1)	4	3.4 (0.9–8.5)	0	0.0 (0.0–3.1)
Total Итого	497	159	32.0 (27.9–36.3)	127	25.6 (21.8–29.6)	12	2.4 (1.3–4.2)	19	3.8 (2.3–5.9)	6	1.2 (0.4–2.6)

 

A comparative analysis of the prevalence of serological markers between age groups revealed no differences in the prevalence of anti-HBs, HIV Ag/Ab, and HBsAg.

A significant difference in the prevalence of anti-HBs between age groups was demonstrated: χ² = 36.25 at p < 0.0001, df = 5. A sequential pairwise comparison revealed no significant differences between age groups 30 years and older. A predominance of individuals with anti-HBs was shown in the 18–29 age group compared to the group comprising individuals aged 30 and older (χ² = 29.0 at p < 0.0001, df = 1).

The χ² function showed statistically significant differences in the distribution of anti-HBcore frequencies among all age groups as a whole (χ² = 20.25 at p = 0.0009, df = 5). In a sequential (from younger to older) pairwise comparison of all 6 groups with each other, adjusted for multiple comparisons, no statistically significant differences between “neighboring” age groups were found. However, a targeted comparison of the oldest age cohort (70+ years) with younger groups showed that the frequency of anti-HBcore detection in the 70+ group was significantly higher than in the 18–29, 30–39, and 40–49 years (p < 0.05, according to Fisher’s criterion), confirming the general trend of a gradual increase in the proportion of individuals with anti-HBcore with age.

The distribution of markers was analyzed. Anti-HCV and anti-HBcore were detected simultaneously in 1.6% of volunteers (8/497, 95% CI 0.7–3.1%). Combinations of HIV Ag/Ab with anti-HBcore and HIV Ag/Ab with anti-HCV were detected in one case each (0.2% (1/497), 95% CI 0.0–1.1%). The distribution of HBV serological markers in the study group is presented in Table 2.

 

Table 2. Distribution of the analyzed hepatitis B (HB) serological markers

Таблица 2. Распределение анализируемых серологических маркеров гепатита В (ГВ)

Hepatitis B Serological Markers Серологические маркеры ГВ	Prevalence Распространенность
	abs. / абс. (n = 497)	% (95% CI) % (95% ДИ)
HBsAg	5	1.0 (0.3–2.3)
Anti-HBs	71	14.3 (11.3–17.7)
Anti-HBc	38	7.6 (5.5–10.3)
HBsAg, anti-HBs	1	0.2 (0.0–1.1)
HBsAg, anti-HBc	2	0.4 (0.0–1.4)
HBsAg, anti-HBs, anti-HBc	4	0.8 (0.2–2.0)
Anti-HBs, anti-HBc	83	16.7 (13.5–20.3)

 

Prevalence of pathogen nucleic acids in the examined group

No HIV RNA was detected in the examined group.

HCV RNA was detected in 5 patients, accounting for 1% (5/497, 95% CI 0.3–2.3%) of the total group and 26.3% (5/19, 95% CI 9.1–51.2%) of HCV-positive individuals. A viral load of more than 1500 IU/ml was detected in 3 samples, which allowed sequencing of virus genome fragments.

Genotyping of 3 HCV samples with sufficient viral load revealed the following genotypes: 1b, 2a and 3a (one sample of each). Although most of the analyzed regions of these samples did not contain significant resistance mutations, the L159F mutation associated with reduced sensitivity to sofosbuvir was identified in the NS5B region of the Dental296 sample (genotype 1b). Furthermore, two substitutions (NS5B C316N and NS3 V170I) were identified in this sample at positions associated with pathogen resistance to dasabuvir, sofosbuvir and voxilaprevir, respectively.

Among the study participants, HBV DNA was detected in 3.8% of cases (19/497, 95% CI 2.3–5.9%). The prevalence of HBV DNA in different age groups is presented in Table 3.

 

Table 3. The prevalence of the HBV DNA across different age groups in the study population

Таблица 3. Распространенность ДНК ВГВ среди обследованных разных возрастных групп

Age group Возрастная группа	Число пациентов Number of patients	ДНК ВГВ / HBV DNA
	abs. абс.	abs. абс.	% (95% CI) % (95% ДИ)
18–29 years / лет	43	2	4.7 (0.6–15.8)
30–39 years / лет	52	2	3.8 (0.5–13.2)
40–49 years / лет	51	1	2.0 (0.0–10.4)
50–59 years / лет	95	4	4.2 (1.1–10.4)
60–69 years / лет	138	8	5.8 (2.5–11.1)
70 + years / лет	118	2	1.7 (0.2–6.0)
Total Итого	497	19	3.8 (2.3–5.9)

 

A sequential pairwise comparison revealed no significant differences in the frequency of HBV DNA between age groups.

It should be noted that only 10 patients had HBV DNA detected simultaneously with HBsAg, accounting for 2.0% (10/497, 95% CI 0.9–3.7%) of the total group, while 1.8% (9/497, 95% CI 0.8–3.4%) of those examined were HBsAg-negative (Table 4).

 

Table 4. The prevalence of the HBV DNA across different age groups in the study population in subjects with and without HBsAg

Таблица 4. Распространенность ДНК ВГВ среди обследованных лиц разных возрастных групп в зависимости от наличия или отсутствия HBsAg

Age group Возрастная группа	Число пациентов Number of patients	HBV DNA, HBsAg+ ДНК ВГВ, HBsAg+		HBV DNA, HBsAg− ДНК ВГВ, HBsAg−
	abs. абс	abs. абс	% (95% CI) % (95% ДИ)	abs. абс	% (95% CI) % (95% ДИ)
18–29 years / лет	43	1	2.3 (0.1–12.3)	1	2.3 (0.1–12.3)
30–39 years / лет	52	0	0.0 (0.0–6.8)	2	3.8 (0.5–13.2)
40–49 years / лет	51	0	0.0 (0.0–7.0)	1	2.0 (0.0–10.4)
50–59 years / лет	95	2	2.1 (0.2–7.4)	1	1.1 (0.0–5.7)
60–69 years / лет	138	7	5.1 (2.0–10.2)	2	1.5 (0.2–5.1)
70 + years / лет	118	0	0.0 (0.0–3.1)	2	1.7 (0.2–6.0)
Итого Total	497	10	2.0 (0.9–3.7)	9	1.8 (0.8–3.4)

 

No significant differences were found between the frequencies of HBsAg-positive and HBsAg-negative HBV infection within age groups.

Based on a comprehensive analysis of genotypes and subtypes, taking into account phylogenetic analysis and analysis using web resources, only genotype D was identified among the detected HBV strains, with the following distribution of subtypes: D1 – 26.3% (5/19, 95% CI 9.1–51.2%), D2 – 36.8% (7/19, 95% CI 16.3–61.6%), D3 – 36.8% (7/19, 95% CI 16.3–61.6%).

Analysis of HBV nucleotide and amino acid sequences revealed multiple amino acid substitutions in the RT (100%, 19/19), SHB (100%, 19/19), Core (100%, 19/19), and preCore (21.1%, 4/19, 95% CI 6.1–45.6%). No samples with drug resistance mutations were identified. The prevalence of amino acid substitutions in the main hydrophilic region (MHR) was assessed (Table 5).

 

Table 5. Mutations identified in the MHR region (aa99–169) within the study group

Таблица 5. Мутации, выявленные в MHR-регионе (aa99–169) в обследуемой группе

Mutations Мутация	Frequency of occurrence in the group Частота встречаемости в группе (n = 19)		Frequency of occurrence in the group HBsAg+ Частота встречаемости в группе HBsAg+ (n = 10)		Frequency of occurrence in the group HBsAg− Частота встречаемости в группе HBsAg− (n = 9)
	abs. абс.	% (95% ДИ) % (95% CI)	abs. абс.	%	abs. абс.	%
Y100С	4	21.1 (6.1–45.6)	0	0	4	44.4
Q101H	1	5.3 (0.1–26.0)	0	0	1	11.1
M103I	4	21.1 (6.1–45.6)	0	0	4	44.4
I110L	1	5.3 (0.1–26.0)	0	0	1	11.1
G112E	1	5.3 (0.1–26.0)	0	0	1	11.1
T113S	19	100.0 (82.4–100.0)	10	100	9	100.0
T114S	19	100.0 (82.4–100.0)	10	100	9	100.0
T116N	3	15.8 (3.4–39.6)	0	0	3	33.3
T118V/A	6	31.6 (12.6–56.6)	5	50	1	11.1
T118R	2	10.5 (1.3–33.1)	0	0	2	22.2
R122K	1	5.3 (0.1–26.0)	0	0	1	11.1
T125M	2	10.5 (1.3–33.1)	0	0	2	22.2
T127P	6	31.6 (12.6–56.6)	2	20	4	44.4
A128V	3	15.8 (3.4–39.6)	2	20	1	11.1
G130R	4	21.1 (6.1–45.6)	0	0	4	44.4
N131T	19	100.0 (82.4–100.0)	10	100	9	100.0
M133L/I	3	15.8 (3.4–39.6)	0	0	3	33.3
Y134H	1	5.3 (0.1–26.0)	0	0	1	11.1
T140I	1	5.3 (0.1–26.0)	0	0	1	11.1
D144E	3	15.8 (3.4–39.6)	0	0	3	33.3
G145R	3	15.8 (3.4–39.6)	0	0	3	33.3
G159A	1	5.3 (0.1–26.0)	0	0	1	11.1

 

Analysis of the spectrum of mutations in the MHR region showed that variability among individuals with HBsAg-negative HBV (n = 9) was due to a significantly higher frequency of specific amino acid substitutions associated with immune escape compared to the group of HBsAg-positive patients (n = 10). In particular, the occult HBV group had significantly more frequent Y100C (44.4% vs. 0%; p = 0.0325), M103I (44.4% vs. 0%; p = 0.0325), and G130R (44.4% vs. 0%; p = 0.0325) mutations were significantly more frequent in the occult HBV group. Other mutations, also known as key escape-variants (T116N, M133L/I, D144E, G145R) were found only in the HBsAg-negative group (33.3% vs. 0% for each), showing a tendency toward higher prevalence (p = 0.08).

In the basal core promoter (BCP) region, the double mutation A1762T/G1764A was detected in 2 samples, accounting for 10.5% (2/19, 95% CI 1.3–33.1%).

Three polymorphic sites were identified in the preCore region, in which amino acid substitutions occurred: L24P, W28*/*W, G29D. In two samples, a combination of W28* + G29D was identified. No significant differences in the frequency of mutations in the preCore region were found between HBsAg-positive and negative samples.

Twenty polymorphic sites were identified in the Core region where amino acid substitutions occurred. The frequencies of mutations identified in the Core region are presented in Table 6.

 

Table 6. Mutations identified in the Core region within the study group

Таблица 6. Мутации, выявленные в Core-регионе в обследуемой группе

Mutations Мутация	Frequency of occurrence in the group Частота встречаемости в группе (n = 19)
	abs. абс.	%	95% CI−, % 95% ДИ−, %	95% CI+, % 95% ДИ+, %
T12S	4	21.1	6.1	45.6
S21Q/A	3	15.8	3.4	39.6
V27I	1	5.3	0.1	26.0
A34T	1	5.3	0.1	26.0
E40D	4	21.1	6.1	45.6
E64DE/D	2	10.5	1.3	33.1
T67N	1	5.3	0.1	26.0
N74A/G/V	19	100.0	82.4	100.0
E77D	1	5.3	0.1	26.0
A80I/T	6	31.6	12.6	56.6
N87S	19	100.0	82.4	100.0
N92H	1	5.3	0.1	26.0
I97F	19	100.0	82.4	100.0
E113D	1	5.3	0.1	26.0
L116I	18	94.7	74.0	99.9
A131P	1	5.3	0.1	26.0
R133KR	1	5.3	0.1	26.0
R151G	1	5.3	0.1	26.0
R174HR	1	5.3	0.1	26.0
S183P	3	15.8	3.4	39.6

 

Discussion

Effective management of biological risks plays a crucial role in the prevention of infections in dentistry, where contact with biological fluids occurs regularly. Particular attention should be paid to assessing the occupational risk of blood-borne infections, which should take into account both the likelihood of contact and the prevalence of specific pathogens. The greatest danger is posed by cases of skin damage with massive contact with blood (e.g., hollow needle sticks), while a smaller but significant risk is associated with blood coming into contact with damaged skin or mucous membranes [14]. According to research conducted in Russia, medical workers encounter glove damage on average 27 times per 100 invasive procedures: in 9 cases, there is a puncture with skin trauma, and in 18 cases, there is no damage to the skin. At the same time, almost half (43%) of surgical procedures are accompanied by various emergency situations [15]. Wearing personal protective equipment (PPE) reduces the risk of infection, but there is evidence that, for example, less than 10% of dentists consistently use protective eyewear [16]. The COVID-19 pandemic has led to stricter compliance with PPE requirements in dental practice [17]. Nevertheless, uncertainty remains regarding the current level of PPE use after the mandatory restrictions were lifted, as many professionals noted that PPE interferes with their work by limiting their field of vision during procedures.

It is also necessary to consider the potential risks of infecting patients through contaminated instruments, which can be prevented by sterilizing instruments. The most common and effective methods are steam pressure sterilization (autoclaving) and dry heat sterilization, each of which has its own advantages and limitations: autoclaving is fast but can cause corrosion and is not suitable for heat-sensitive instruments, while dry heat does not cause corrosion but requires high temperatures and equipment calibration. Alternative methods, such as chemical vapor and ethylene oxide sterilization, are used less frequently due to specific requirements (the necessity for pre-drying, toxicity) and are mainly used for processing heat-sensitive materials. The choice of method depends on the type of instruments, safety requirements and practical feasibility [18], and the quality of sterilization determines the likelihood of patient infection.

Important factors in the infection of both dentists and their patients include the pathogen’s ability to survive on surfaces, the infectious dose, and the viral load in each case. Epidemiological studies show that the risk of HIV infection after a needle stick with a contaminated instrument is 0.3%, and when infected blood comes into contact with mucous membranes, it is 3.3 times lower (0.09%) [19]. The risk of infection is significantly higher in the case of HCV: when HCV RNA is detected, the probability of transmission reaches 10%, which is 3–5 times higher than the corresponding figure (1.8–3.1%) for cases with undetectable levels [15]. The risk of HBV transmission can reach 40% [15, 20], but in this case, it is also necessary to consider the probability of infection from individuals with HBsAg-negative HBV with an undetectable viral load, since the infectious dose of HBV is only 16 copies of the virus, or 3.5 IU/mL [21].

As mentioned above, in order to assess the occupational risk of infection and the risk of pathogen transmission between patients, it is important to consider not only the likelihood of contact with potentially infected biological fluids and contaminated instruments, but also the prevalence of blood-borne infections among individuals seeking dental care.

However, the search for literature in open sources did not reveal any similar studies in St. Petersburg or in the Russian Federation as a whole, which underscores the novelty of the data presented. This is especially relevant given the significant number of publications indicating a high prevalence of dental pathology among both HIV-infected individuals [22], and individuals with parenteral viral hepatitis [23]. Studies conducted in other countries demonstrate a wide range of indicators, strongly dependent on the region and the overall epidemiological situation [24–26], which makes local monitoring relevant.

In the group examined, the prevalence of HIV Ag/Ab was 1.2%, with 2 samples confirmed by immunoblotting, accounting for 0.4% of the total group. HIV RNA was not detected. Apparently, two patients with confirmed HIV infection received antiretroviral therapy, which significantly reduces the likelihood of pathogen transmission.

In the Russian Federation, the overall prevalence of HIV infection is about 0.78%, with this figure reaching 1.42% among the population aged 15–49 years, and the highest values (1.27–2.73%) recorded in the 30–49 age group [27]. Our data are generally consistent with these indicators, although the frequency of HIV Ag/Ab detection in our study slightly exceeds the all-Russian data, but the frequency of confirmed HIV infection corresponds to the overall prevalence of the pathogen in the population. The prevalence of HIV in the surveyed group was, as expected, lower than among persons held in pretrial detention centers (23.19%) [28], but higher than among healthcare workers (0%) [29].

The anti-HCV seroprevalence in the examined group was 3.8%, which is higher than both the population level (1.7%) and the level among healthcare workers (1.17%) [29], but lower than the prevalence of the marker among prisoners (60.14%) [32], and among HIV-infected individuals (18.87%) [30]. At the same time, the prevalence of active infection, determined by the presence of HCV RNA, does not exceed 1%, which reflects the incidence of hepatitis C in the Northwestern Federal District (NWFD) (49.2 cases per 100,000 population) [31].

However, nucleotide sequence analysis conducted on a limited sample of three specimens allowed for the identification of HCV genotypes 1b, 2a, and 3a, which are characteristic of the Russian Federation and St. Petersburg [7, 31].

The detection of the L159F mutation in the NS5B region of the Dental296 sample (genotype 1b) is of particular interest because, according to the literature, this substitution is associated with a decrease in the sensitivity of the virus to sofosbuvir, a key drug in pan-genotypic treatment regimens. According to research, L159F belongs to the so-called “polymorphic” mutations, which can also occur in untreated patients, but their presence requires careful monitoring of the effectiveness of therapy. Two other substitutions of interest that have long been considered atypical and clinically insignificant are C316N in NS5B and V170I in NS3, which were identified in the same sample. Although the C316N mutation is rare in natural HCV populations (frequency less than 1% according to the EU HCV Resistance Database), it is known as a marker of resistance not only to sofosbuvir but also to dasabuvir, a component of combination treatment regimens [32]. This is because positions NS5B 316, 414, 448, 553 and 556 represent the binding pocket of dasabuvir [33]. Similarly, according to clinical observations, a substitution at position 170 in the NS3 protease may reduce the efficacy of voxilaprevir, a second-generation protease inhibitor. It should be noted that the V170I substitution has been experimentally shown to be associated with reduced viral susceptibility to boceprevir and simprevir [34]. The combined presence of these three mutations in a single clinical isolate, especially in a person who has not previously received PPD treatment, is particularly concerning, as it may potentially limit therapeutic options. It is important to note that, according to Russian studies [31], genotype 1b remains dominant in the Russian Federation, which makes monitoring of such mutations particularly relevant for domestic clinical practice. The frequency of the above-described HCV mutations in the Northwestern Federal District is 1.6–2% for L159F; for C316Y/N, it ranges from 2% among treatment-naive individuals to 9.1% among individuals with virological breakthrough during therapy; V170I was not detected [35]. The data obtained emphasize the need to continue research on the prevalence of resistant HCV strains in the region and to develop personalized approaches to the treatment of chronic hepatitis C.

The prevalence rates of HBV markers are of particular epidemiological significance. High levels of HBsAg (2.4%) and anti-HBcore (25.6%) were recorded in the study group, indicating a significant proportion of individuals with current or past infection and a relatively low anti-HBs rate (32.0%), reflecting insufficient post-infectious or post-vaccination immunity in the population. In Russia, the epidemiological situation with regard to viral hepatitis shows contradictory trends: despite a steady decline in the incidence of acute forms, chronic hepatitis B remains widespread. In 2024, the rate of newly diagnosed cases of chronic hepatitis B reached 9.41 cases per 100,000 population (13,770 cases), which is 11.4% higher than in 2023 (8.45 per 100,000), but 12.7% lower than in 2015 (10.78 per 100,000)^5,6. The situation in St. Petersburg is of particular concern, where the incidence of chronic hepatitis B consistently exceeds the Russian average^5,6. Although there has been a gradual decline in chronic hepatitis B incidence rates since 1998, their persistently high levels against the backdrop of low HCV detection rates indicate the existence of a stable reservoir of infection and ongoing active transmission of the virus. The high frequency of HCV markers detected in this study among dental patients does not allow medical procedures to be excluded as a significant route of transmission. This seems particularly important, as the detected levels of HBsAg (2.4%) and anti-HBcore (25.6%) are significantly higher than the prevalence of these markers in the general population (1.3% and 11.3%, respectively) and among healthcare workers (0.58% and 10.53%) [29], but lower than among individuals in penitentiary institutions (3.2% and 37.68%) [28].

The data obtained revealed statistically significant differences in the frequency of anti-HBs and anti-HBcore among different age groups. A characteristic pattern was observed: anti-HBs levels are higher in younger age groups, while anti-HBcore, on the contrary, is more commonly found in older individuals. Although the study is limited by uneven distribution and a small sample size across age groups, the pattern identified suggests that, if the study is expanded, a more pronounced age dependence will be observed — a progressive increase in the frequency of anti-HBcore and a decrease in anti-HBs levels with increasing age of the subjects. In Russia, mass vaccination against HBV began relatively late—only in 1997 was this type of immunization included in the national vaccination schedule. Four years later, in 2001, a vaccination program for 13-year-old adolescents was launched, covering 96.5% of the target group, while among adults from professional risk groups, vaccination coverage was only 10–40%. A significant expansion in hepatitis B vaccination coverage occurred in 2006 with the launch of the national Health Project, which provided for additional immunization of adults aged 18 to 55. In view of the above, it is evident that the identified patterns are associated with the introduction of stages of vaccination against HBV, as well as with the fact that the probability of encountering infection increases statistically with age. Indirect confirmation of this assumption is provided by the similar age dynamics of anti-HBs and anti-HBcore (with an increase in detection frequency in the 60–70+ age groups), indicating that the increase in anti-HBs levels in older individuals most likely reflects post-infectious immunity rather than the effects of vaccine prophylaxis. This correlation between markers is an important argument in favor of their infectious origin in these age categories.

A significant proportion of individuals with a combination of HBsAg + anti-HBs + anti-HBc (0.8%) and anti-HBs + anti-HBc (16.7%) markers is noteworthy. Analysis of the results obtained allows us to identify four possible explanations for the observed serological combinations: the presence of active HBV infection at the time of examination; a complex of post-infectious antibodies; vaccination of previously infected individuals without prior screening for HBV markers; infection of vaccinated patients with HBV strains containing escape mutations (EM). Each of these options may contribute to the formation of the identified serological profiles. These data emphasize the importance of a comprehensive assessment of infection markers when interpreting the results of serological studies.

A high frequency of HBV DNA (3.8%) was observed, including in the absence of HBsAg (1.8%). Interestingly, the frequency of HBsAg-positive cases was higher than in the general population and among blood donors (0.4% each) and similar to the frequency among pregnant women (1.9%) [36]. At the same time, the prevalence of HBsAg-negative (occult) HBV is similar to that in the general population (1.7%), but lower than among pregnant women (2.8%) and blood donors (2.7%) [36]. When assessing the prevalence of HBV DNA, no significant differences in the frequency of HBV DNA were found between age groups, regardless of the presence or absence of HBsAg.

Molecular genetic analysis showed that the HBV strains studied belong to genotype D, which corresponds to the current epidemiological situation in the Russian Federation and St. Petersburg, where this genotype is predominant. Although, according to publications [31], cases of infection with genotypes A and, in rare cases, C are also registered in the region, these variants were not identified in this study, which is apparently due to the limited number of HBV DNA-positive samples in the cohort under study.

Analysis of HBV sequences did not reveal any mutations associated with drug resistance. This observation can be explained by two main factors: firstly, the absence of antiviral therapy in the patients examined, which excludes selective pressure contributing to the emergence of resistant strains; second, the low natural prevalence of viral variants with similar mutations among the untreated population in this region. This epidemiological picture is consistent with known data that resistance mutations predominantly arise and become established in the virus population under the influence of specific therapy, occurring relatively rarely among untreated individuals [36].

The most significant result of the study was the almost complete penetrance of escape mutations in the cohort studied. In all analyzed HBV samples of the only detected genotype D, amino acid substitutions were detected in three structurally significant positions – T113S, T114S, and N131T, which indicates their potential significance in the characteristic molecular genetic features of this genotype. Although mutations at these positions have been described by some authors as being associated with immunological evasion, it is clear that the polymorphic variants identified are characteristic of genotype D and either do not relate to EM or are indirect evidence of a greater predisposition of HBV genotype D to develop HBsAg-negative chronic hepatitis B. Anti-HBs IgG antibodies induced by HBV vaccination is primarily directed at recognizing the hydrophilic domain of HBsAg corresponding to the conformational antigenic determinant “a” (amino acid positions 124–147). In the course of this study, a spectrum of mutations was identified, including known genetic variants associated with the phenomenon of evasion of vaccine-induced immune response, as well as those capable of disrupting HBsAg secretion and leading to reinfection in liver tissues despite protective anti-HBs titers: T125M, T127P, A128V, G130R, N131T, M133L/I, Y134H, T140I, D144E, G145R [37, 38]. Furthermore, a significant number of mutations detected in the S gene in the study group are associated with the development of occult chronic hepatitis B, including outside the “a” determinant: Y100C, Q101H, M103I, D144E, G145R [37–39]. The significance of these mutations is clearly confirmed in this study, including by the fact that most of the above mutations are identified in HBsAg-negative HBV, including in those with anti-HBs. According to available data, the average frequency of mutations in the HBsAg gene varies from 11% in North American populations to 47% among patients with chronic hepatitis B in South Korea, while in the MHR region, the frequency of mutations demonstrates a genotype-dependent nature, ranging from a minimum of 57.5% for genotype A to 100% for genotype D [40], which corresponds to our results.

In the studied group, the double mutation BCP A1762T/G1764A was detected in 10.5% of cases, which is clinically significant given its proven association with the development of liver cirrhosis and hepatocellular carcinoma (HCC) [41]. This mutation has a multifactorial effect on the pathogenesis of HBV infection, including a decrease in HBeAg production due to the formation of a stop codon, increased pregenomic RNA transcription, and changes in the intracellular localization of HBcAg, which contributes to the development of an active inflammatory process. Experimental studies demonstrate that this mutation causes significant changes in cell cycle regulation by activating S-kinase protein 2 while simultaneously suppressing the p21 inhibitor [42], and may also serve as an early marker of carcinogenesis, being detected a decade before the development of HCC [43]. Most mutations in the preCore/Core region are point substitutions that primarily affect HBeAg levels and viral load, and in the Core region, they are mainly localized in immunoreactive domains (MHC classes I and II), which can potentially modulate the course of the disease [44].

The study identified clinically significant mutations in the preCore region of HBV in 21.1% of patients. The dominant G1896A (W28*) substitution, which disrupts HBeAg synthesis [36], is particularly characteristic of genotypes A/D [45]. The G1899A mutation (10.5% of cases in the cohort studied) also contributes to the progression of fibrosis and HCC [41, 42]. The W28*W and W28*Y variants indicate a possible accumulation of W28* during chronic infection.

The localization of amino acid substitutions in the Core region is of particular interest, as many mutations affect strategically important immunodominant regions of HBcAg. These are primarily CD4⁺ T-cell epitopes (aa1–20, 50–69, 81–105, 117–131, 141–165), cytotoxic CD8⁺ T-cell epitopes (aa18–27, 88–96, 130–140, 141–151), and B-cell epitopes (aa74–89, 107–118, 127–138) [41]. A significant proportion of the mutations identified (Table 6) occur in these immunologically active regions, creating the molecular conditions for the virus to evade the immune response. Such changes can disrupt antigen processing and presentation, reduce binding affinity to T- and B-cell receptors, and ultimately contribute to viral persistence and chronic hepatitis B progression. The amino acid substitutions identified in the Core region demonstrate two key pathogenic effects: an impact on viral persistence and the clinical course of infection. Structural and functional changes induced by the A131P and R133KR mutations at positions 113–143 lead to modification of antigenic determinants and destabilization of the nucleocapsid, which provides a selective advantage to immune-evading variants of the virus. At the same time, genetic markers of progressive liver damage have been identified, including those located in B-cell epitopes E77D, A80T, and L116I/V/G (polymorphism at this position is present in almost all samples), as well as in T-cell epitopes E64D. These polymorphic variants are associated with histopathological changes such as cirrhosis and HCC [41, 42].

Despite significant heterogeneity of data due to interpopulation differences (including variability of viral genotypes, patient ethnicity, immunological status, and presence of coinfections) and methodological discrepancies between studies [41], a stable cluster of amino acid substitutions in the preCore/Core regions of HBV has been identified, demonstrating a statistically significant association with progressive fibrogenesis and HCC. Mutations that modulate HBeAg expression and have been verified as independent predictors of cirrhosis and HCC development are of particular diagnostic significance, allowing them to be considered as promising molecular markers for risk stratification and predictive diagnosis of adverse outcomes of HBV infection.

Mutations in key immunodominant regions of viral proteins can significantly disrupt immune recognition processes, creating conditions for the development of persistent HBV infection with a number of clinically significant consequences. In particular, such amino acid substitutions contribute to the formation of HBsAg-negative forms of HBV and also lead to increased variability in functionally significant regions of the viral genome. These changes underlie the mechanisms by which the virus evades immune control, which ultimately determines the chronicity of the infectious process and its progressive course [46].

The identified variability of the studied HBV genome regions corresponds to known data on the significant natural heterogeneity of the genotype D virus, which is associated with the chronicity of the infectious process, the progression of pathology, and the formation of a latent form of HBV.

Conclusion

The high risk of contact with biological fluids during dental procedures, combined with a significant proportion of patients with undiagnosed forms of viral hepatitis B, C, as well as HIV infection, requires the unconditional use of PPE, mandatory vaccination of medical personnel against HBV, and, in the event of an emergency, comprehensive post-exposure prophylaxis, including a thorough epidemiological history, assessment of the infectious status of the source patient, analysis of the characteristics of contact, and determination of the victim’s immunological susceptibility to HBV.

This problem is particularly relevant given the high prevalence among dental patients of the HBsAg-negative form of HBV, associated with the circulation of viral variants containing escape mutations. The detection of isolates characterized by the simultaneous presence of mutations leading to diagnostic errors in the detection of HBsAg, reduced vaccine efficacy, virus reactivation and disease progression poses a serious threat to the healthcare system and requires further in-depth research.

¹ UNAIDS. Global HIV & AIDS statistics – Fact sheet. Доступно по: https://unaids.org/en/resources/fact-sheet

² WHO. Hepatitis B. Key facts. Available at: https://who.int/news-room/fact-sheets/detail/hepatitis-b

^{3 3}WHO. Hepatitis C. Key facts. Available at: https://who.int/news-room/fact-sheets/detail/hepatitis-c

⁴ WHO. Regional strategic framework for vaccine-preventable diseases and immunization in the Western Pacific 2021-2030; 2022. Available at: https://who.int/publications/i/item/9789290619697

⁵ State report «On the state of sanitary and epidemiological well-being of the population in the Russian Federation in 2024». Moscow; 2025.

⁶ State report «On the state of sanitary and epidemiological well-being of the population in the Russian Federation in 2023». Moscow; 2024.

About the authors

Yulia V. Ostankova

St. Petersburg Pasteur Institute

Email: shenna1@yandex.ru
ORCID iD: 0000-0003-2270-8897

Cand. Sci. (Biol.), Senior Researcher at the Laboratory of Molecular Immunology, Head of the Laboratory of Immunology and Virology HIV Infection

Russian Federation, 197101, St. Petersburg

Elena N. Serikova

St. Petersburg Pasteur Institute

Email: elena.donetsk.serikova@mail.ru
ORCID iD: 0000-0002-0547-3945

Researcher, Laboratory of Immunology and Virology HIV Infection

Russian Federation, 197101, St. Petersburg

Alexandr N. Schemelev

St. Petersburg Pasteur Institute

Email: tvildorm@gmail.com
ORCID iD: 0000-0002-3139-3674

PhD, Junior Researcher, Laboratory of Immunology and Virology HIV Infection

Russian Federation, 197101, St. Petersburg

Ekaterina V. Anufrieva

St. Petersburg Pasteur Institute

Email: kate.an21@yandex.ru
ORCID iD: 0009-0002-1882-529X

Junior Researcher, Laboratory of Immunology and Virology HIV Infection

Russian Federation, 197101, St. Petersburg

Elena B. Zueva

St. Petersburg Pasteur Institute

Email: ezueva75@mail.ru
ORCID iD: 0000-0002-0579-110X

PhD, Biologist at the Department of Diagnostics of HIV Infection and AIDS-Associated Diseases

Russian Federation, 197101, St. Petersburg

Olga S. Kreidik

St. Petersburg State Budgetary Institution of Health «City Polyclinic No. 38»

Email: Kreidik@mail.ru
ORCID iD: 0009-0000-2061-7073

Dentist-surgeon, Аdult dental department

Russian Federation, 191015, St. Petersburg

Marina B. Kusevitskaya

St. Petersburg State Institution of Health «City Clinical Hospital No. 31»

Email: mbkus@mail.ru
ORCID iD: 0000-0002-7196-2595

PhD (in Medicine), obstetrician-gynecologist of the Department of Operative Gynecology

Russian Federation, 197110, St. Petersburg

Leonid Ya. Kusevitskiy

First St. Petersburg State I. Pavlov Medical University

Email: stomdoc@mail.ru
ORCID iD: 0000-0001-5739-5179

PhD, MD (Medicine), dentist-orthopedist of the highest qualification category, Professor of prosthodontics and materials science with orthodontics course

Russian Federation, 197022, St. Petersburg

Areg A. Totolian

St. Petersburg Pasteur Institute; First St. Petersburg State I. Pavlov Medical University

Author for correspondence.
Email: totolian@pasteurorg.ru
ORCID iD: 0000-0003-4571-8799

Academician of the Russian Academy of Sciences, PhD, MD (Medicine), Professor, Head at the Laboratory of Molecular Immunology, Director, head Department of Immunology

Russian Federation, 197101, St. Petersburg; 197022, St. Petersburg

References

Supplementary files