An estimated 325 million of people, in 2015, were chronic carriers of hepatitis B virus (HBV) or hepatitis C virus (HCV) worldwide [1, 2]. The endemicity is described by the prevalence of the hepatitis B surface antigen (HBsAg) [3]. Globally around 257 million are chronically infected with HBV, and 890,000 die each year from HBV-related hepatic complications [1, 4–6]. Studies have estimated that 30% of cirrhosis and 53% of liver cancer in the world are associated with chronic HBV infection, and 15–40% of patients with chronic HBV infection will develop cirrhosis, liver failure, or hepatocellular carcinoma (HCC) in their lifetime [1, 3]. It is estimated that 70–95% of the adult population in sub-Saharan African countries have been exposed to HBV, and HBsAg sero-prevalence ranges from 6–20% [1–3, 5]. For example, a systematic review and meta-analysis study conducted in Cameroon showed that the overall prevalence of HBV was 11.2% [7], a population-based study of all ages reported an HBsAg prevalence of 17.6% in Northern Uganda which increased with increasing age [8].

HBV is easily transmitted from mother to child, usually at the time of delivery. Without immuno-prophylaxis, the risk of HBV infection among infants born to women who are both HBsAg-positive and HBeAg-positive (a marker of high virus titers) is approximately 85–100%, and 90–95% of these infants become chronically infected with HBV [9, 10]. Knowledge of the prevalence of chronic HBV infection among pregnant women is critical to understand the epidemiology of the disease. Studies and systematic reviews have found that the prevalence of HBsAg among pregnant women in a collection of African nations varies from 2.4–16% [11]. For example, a study conducted among Sudanese pregnant women found a prevalence of 5.6%; [12] similar studies estimated a prevalence of 8.2% in Nigeria [13], 6.3% in Tanzania [14], 8.0% in Mali [15].

HBV and HIV have similarities by exposure to infectious blood and body fluids, but not all the same modes of transmission. Unlike HIV, HBV is not transmitted by breastfeeding, furthermore, child to child transmission is common for HBV but not for HIV and there is evidence suggesting that HBV is more infectious than HIV [2]. There are higher concentrations of HBV in body fluids of persons with acute and chronic HBV infection than among persons with HIV infection. This may possibly explain why HBV might be more infectious than HIV. It is estimated that the prevalence of HBV among HIV infected patients worldwide is 10% and in countries with high HBV prevalence the estimates of HBV infection among HIV infected persons can reach up to 31.3% [16]. Co-infection of HBV and HIV among pregnant women attending ANC in Zambia was 31.3%, 9.0% in Ivory Coast, and 4.9% in Uganda [15]. In North Region of Cameroon, the prevalence of HIV and HBV co-infection was 1.5% [16]. HBV-related liver diseases are more progressive in HIV co-infected patients than in patients with HBV infection alone [1].

Limited information is available on the prevalence of HBV among pregnant women in Rwanda. A study done by Pirillo and colleagues found that the prevalence of HBV among pregnant women in Rwanda was 2.4% [17]. However, that study was not nationally representative. Moreover, a better understanding of factors associated with HBV-HIV co-infection would be beneficial to any prevention programmes aiming to curb the spread of both infections. Therefore, the aim of this study was to estimate the prevalence of chronic HBV infection among pregnant women attending ANC in Rwanda and to determine risk factors associated with HBV and HIV co-infection.

Nationally, 13,292 pregnant women were enrolled in the survey and 13,121 of them had HBsAg test results. For different reasons (refusal, insufficient blood sample), 0.6% of participants were not tested for HBsAg. Figure 2 provides further insights into the test result numbers using the flow chart. The overall prevalence of HBsAg was 3.70% (95% CI: 3.8, 4.0%). There were no significant differences in HBsAg prevalence among all socio-demographic characteristics assessed (Table 1). Of the 486 women who tested HBsAg-positive, only 178 (37.0%) samples were tested for HBeAg and 40 samples (22.5%) tested HBeAg-positive. The prevalence of HBcAb-IgM among HBsAg-positive women was 2.1% suggests that there was 2.1 recent HBV infections per every 100 HBsAg positive pregnant women. Of the 486 women who tested HBsAg-positive, 20 women (4.1% [95% CI: 2.5–6.3%]) were also infected with HIV. There was no observed HBV-HIV co-infection among pregnant women in ‘salaried’ occupation, but this co-infection was 4.3% [95% CI: 2.6–6.5%] among pregnant women in ‘not salaried’ occupation. HBV-HIV co-infection was more than three times higher among women in urban areas (6.6% [95% CI: 6.0–7.2%]) compared to women in rural areas (1.9% [95% CI: 0.6, 4.5%]). For other socio-demographic characteristics, there is no significant difference in HBV-HIV co-infection prevalence (Table 2).

Characteristics	Total	HBsAg Positive			P-value
	N	n	%	95% CI
Overall	13,121	486	3.7	[3.4, 4.0]
Age group
15–24 yrs	4719	182	3.9	[3.4, 4.5]	0.528
25–49 yrs	8326	303	3.6	[3.2, 4.0]
Marital Status
In union	11,366	424	3.7	[3.4, 4.1]	0.993
Not in union	1664	62	3.7	[2.9,4.8]
Education level
Illiterate/primary	11,536	425	3.7	[3.4, 4.1]	0.517
Secondary/University	1466	59	4.0	[3.1, 5.2]
Occupation
Salaried	500	15	3.0	[1.7,4.9]	0.387
Not salaried	12,492	468	3.7	[3.4, 4.0]
Residence
Rural	6547	257	3.9	[3.4, 4.4]	0.180
Urban	6574	229	3.5	[3.1, 3.9]
Pregnancies
≤ 2 pregnancies	6946	252	3.6	[3.2,4.1]	0.637
> 2 pregnancies	5837	221	3.8	[3.3,4.3]

	HIV infection among HBsAg-positive pregnant women			Bivariate mixed-effect logistic model	Multiple mixed-effects logistic model
		n	%	Odds ratio (95% CI)	Adjusted odds ratio (95% CI)
Overall	486	20	4.1
Age group
25–49 years	303	10	3.3	1	1
15–24 years	182	10	5.5	1.82 (0.71 – 4.55)	6.91 (1.77 – 27.01)
Marital status
In union	424	17	4.0	1
Not in union	62	3	3.8	1.19 (0.25 – 5.66)	NA
Education level
Illiterate/primary	425	17	4.0	1
Secondary/University	59	3	5.1	1.03 (0.27 – 3.91)	NA
Occupation
Salaried	15	0	0.0
Not Salaried	468	20	4.3	PFP	NA
Residence
Rural	257	5	1.9	1	1
Urban	229	15	6.6	3.28 (1.02 – 10.53)	4.34 (1.15 – 16.43)
Pregnancies
< = 2 pregnancies	252	8	3.2	1	1
> 2 pregnancies	221	11	5.0	1.60 (0.61 – 4.19)	6.88 (1.71 – 27.77)
RPR results
RPR negative	475	16	3.4	1	1
RPR positive	11	4	36.4	17.28 (3.98 – 74.93)	24.89 (5.04 – 122.89)

CI confidence interval, RPR rapid plasma reagin

PFP predicts failure perfectly

NA not applicable refers to factors with p-value ≥0.25 at the bivariate level which were not considered when developing the Multivariable model. The variables listed in this table are the variables that were included in the bivariate analyses

In the multivariable survey logistic regression model, age, marital status, education level, residence, number of pregnancies, and RPR status were considered as potential risk factors for HBV-HIV co-infection. After adjusting for potential confounders, younger women 15–24 years were more likely to have HBV-HIV co-infection compared to older women aged 25–49 years (aOR = 6.9; (95% CI: 1.8–27.0). Women residing in urban areas were more likely to have HBV-HIV co-infection compared with those residing in rural areas (aOR = 4.3; 95% CI: 1.2–16.4). Women with more than two pregnancies were more likely to have HBV-HIV co-infection (aOR = 6.9; 95% CI: 1.7–27.8). Women who tested RPR-positive were more likely to be infected with HBV-HIV co-infection (aOR = 24.9; 95% CI: 5.0–1224.9) (Table 2).

The prevalence of HBsAg among pregnant women was similar in all sentinel sites surveyed, and the overall HBsAg prevalence (3.7%) is lower than the prevalence found in studies conducted in neighboring countries [8]. There was no significant variation in HBsAg prevalence in different socio-demographic characteristics. HBV prevalence was slightly higher among women tested positive for HIV (i.e., HBV-HIV co-infected women). Despite the low number of HBV-HIV co-infections in our sample, our model revealed multiple variables that were statistically distinguishable between co-infected women and those without the co-infection. These variables could provide insights into prevention strategies to be adopted for this population. Most importantly, the prevalence of HBV-HIV co-infection among younger women may highlights the need to maximize prevention of perinatal and early childhood transmission of HBV by achieving and sustaining high hepatitis B vaccination coverage among children, and by introducing universal hepatitis B birth dose vaccination. On the other hand, the low proportion of chronic HBV infections among HIV-infected patients shows that the hepatitis B vaccination strategies should be largely enforced independently of HIV prevention strategies.

Rwanda has a lower HIV prevalence than many of its immediate neighbours [8, 18, 19]. Potential reasons for the success of Rwanda’s HIV healthcare programme include its mature national programme and universal healthcare; however, reason for success also includes non-transferable qualities, such as its population density [20–22]. HIV prevalence follows the same trend HIV/HBV co-infection in association with higher number of pregnancies, not salaried occupation status, residing in urban residence, and syphilis infection.

We found that the proportion of pregnant women with HIV among HBsAg-positive pregnant women was 4.1%. This co-infection proportion was similar to the HBV and HIV co-infection prevalence among pregnant women in other sub-Saharan African countries: in Nigeria it was 4.2% [23], 4.9% in Uganda [8] and 5.3% from one study done in South Africa [24], lower than the co-infection found in Cameroon (1.5%) [16]. The prevalence of HBV and HIV co-infection in our survey was much lower than the HBV and HIV co-infection found among pregnant women in Zambia (31.3%) [2], Ivory Coast (9.0%) [2], and in one city of Ethiopia (19.0%) [25]. but higher than the HBV and HIV co-infection found by one study in South-Africa (3.1%) [15] In addition to age, our study identified areas of residence as a possible factor of the co-infection –suggesting that effort to identify co-infection persons should start in urban areas and expand out into the rural areas. Having more than two pregnancies indicates the perhaps higher sexual activity. Similarly, in Nigerian women having two or above pregnancies was also identified as risk factor associated with HBV and HIV co-infection [11]. Having a positive RPR test in pregnant women indicates also a possible sexual risky behavior for HIV and HBV infections.

Our survey has a variety of strengths and limitations. Among its strengths, there was a very high participation rate (98.7%). The most important limitation to our study was the lack of randomness in the selection of the sites included in the survey, however, all sentinel sites were selected to represent the country. Given the small number of sentinel sites, we preferred matching over factors we felt were important to keep balanced namely location and whether sites were urban. Results from this survey can therefore not be generalized to all pregnant women in the country. At the time of the survey, there was a shortage of HBeAg reagents; many HBsAg positive samples were not tested for HBeAg. It would therefore not be possible to extrapolate the associations between HBeAg results and any of the assessed characteristic to the entire study population. The HIV prevalence in this manuscript was based on EIA screening test and EIA confirmatory test as per the national guidelines at that time. Based on the current knowledge, the use of two-test EIA with an EIA confirmatory test would result in a high level of false-positives due to the highly sensitive nature of the fourth generation EIAs that were used in the survey. Rwanda is a country with a small population and low HIV prevalence, therefore the problem of false-positives was not excessive. An EIA confirmatory test was also used to confirm HIV rapid test. A Western Blot test would have added value as a tie breaker. Further studies with more representative sample size to confirm what was observed and depict more information are needed.

HBV infection is a public health problem among pregnant women in Rwanda, as is HBV-HIV co-infection. Understanding that HBV and HBV-HIV co-infection may be more prominent in younger women from urban residences will help strengthen HBV prevention, screening and treatment, which is crucial to the surveyed population of pregnant women in Rwanda. HBV screening in all pregnant women attending ANC services is recommended, especially HIV-positive pregnant women to avoid maternal transmission. The implementation of HBV birth dose vaccination in Rwanda could decrease the burden of HBV among youth.