Hepatitis B and HIV coinfection in Northern Uganda: Is a decline in HBV prevalence on the horizon?

BACKGROUND: The available data concerning hepatitis B virus (HBV) infection in Uganda are limited, particularly in the case of people living with HIV/AIDS (PLWH). HBV is not routinely tested when starting antiretroviral therapy (ART). We aimed to determine the prevalence, the correlates of the risk of HBV infection, and the association with outcomes of ART among PLWH attending a busy HIV clinic in a referral hospital in Northern Uganda. PATIENTS AND METHODS: From April to June 2016, a random sample of 1000 PLWH attending the outpatients' clinic of St. Mary's Hospital, Gulu, Uganda were systematically selected to undergo a rapid hepatitis B surface antigen (HBsAg) test after administering a questionnaire in this cross-sectional study. HIV care parameters were obtained from client files. Multivariate logistic regression and general linear model were used for the analysis.
RESULTS: 950 of the 985 evaluable patients (77% females; mean age 42.8 years) were receiving ART. The overall prevalence of HBsAg was 7.9% (95% confidence interval [CI] 6.2-9.6%), and was significantly lower among the females (6.8% vs 11.7%; p = 0.020). The factors independently associated with higher HBV infection were having lived in an internally displaced persons' camp (adjusted odds ratio [aOR] 1.76, 95% CI 1.03-2.98; p = 0.036) and having shared housing with HBV-infected people during childhood (aOR 3.30, 95% CI 1.49-7.32; p = 0.003). CD4+ T cell counts were significantly lower in HBV patients (p = 0.025), and co-infection was associated with a poorer CD4+ T cell response to ART (AOR 0.88; 95% CI 0.79-0.98; p = 0.030).
CONCLUSIONS: The observed prevalence of HBV among the PLWH may be underestimated or a signal of HBV decline in the region. The factors favouring horizontal HBV transmission identified suggest extending HBV screening and vaccine prophylaxis among PLWH.

Introduction

Approximately 7.4 percent of persons living with HIV (PLWH) worldwide are infected with hepatitis B virus (HBV) with considerable geographical variation depending on overall HBV prevalence in the country [1, 2]. Prevalence of HIV-HBV coinfection is 4–6% in countries with low HBV endemicity and 10–25% in those with intermediate and high endemicity [3]. In areas with low HBV endemicity, both viruses are more often acquired in adulthood through sexual and percutaneous contacts, whereas in hyperendemic horizontal and mother-to-child transmission of HBV predominate [3, 4]. In Africa, the World Health Organisation (WHO) estimates a low/intermediate prevalence of HBV in northern Africa (2–4%), high/intermediate prevalence in Sudan, South Sudan, and central, eastern and southern Africa (5–7%), and high prevalence above eight percent in Chad, Cameroon, and western Africa [1, 2]. However, there is paucity of HIV-HBV co-infection data, although reports show that up to 36% of some cohorts of people living with HIV/AIDS (PLWH) have chronic hepatitis B infection [5].

Bwogi et al. [6] reported an 8.3% prevalence of co-infection in Uganda in 2005, while the estimated prevalence of general HIV was 7.4% [7] in 2015 and 6.2% in 2017 [8]. HBV prevalence was estimated at 10.3% [6] in 2005 and 4.1% in 2017 [8]. If these data are accurate, this would signal a large reduction in HBV prevalence in Uganda. Uganda however has wide regional variations in the prevalence of both HIV and HBV infections [6-8]. In Northern Uganda, the reported prevalence of HIV is 3.1–7.2% and that of HBV is 6–12% in the Acholi subregion of Northern Uganda [8] though earlier figures showed 18.5–23,9%. The same study showed a lower HBV prevalence in Southern Uganda of 4.7–8% [6, 8]. Furthermore, as Northern Uganda was the theatre of a civil war from 1989 to 2006 [9], civilians were compelled to seek refuge in camps where hygiene and living conditions were often poor [10]. In the Gulu region of where this study was carried out, an earlier population based survey in 2010 determined the prevalence of HBV infection to be 17.6% (95%CI 14.9–20.3) [11] with no documentation of HIV status. There is insufficient data to show that regional differences in HIV and HBV prevalence correlate with the prevalence of HBV-HIV coinfection.

HBV is a major cause of morbidity and mortality in PLWH [5, 12–14]. PLWH are at higher risk of occult HBV infection than their HIV-negative counterparts and have six times higher risk of developing chronic hepatitis after acute HBV infection [4, 5, 13, 15]. Moreover, co-infected persons more rapidly develop liver fibrosis, cirrhosis, end-stage liver disease and hepatocellular carcinoma (HCC) [3–5, 13, 15]. Coinfected patients have lower CD4+ T cell counts which respond more slowly to antiretroviral therapy (ART) [16-18], and are more likely to develop immune reconstitution inflammatory syndrome (IRIS) and drug-induced liver damage [13, 19] However, CD4+ T cell count rescue in response to ART is better in HIV/HBV patients who develop hepatitis B surface antibodies (HBsAb) than in those who fail to clear HBV [20].

Given the similarities of the enzymatic pocket of HIV reverse transcriptase and HBV DNA polymerase, some HIV-reverse transcriptase inhibitors can target both viruses [2, 15, 21]. However, underdiagnosis of HBV infection can lead to a failure to start PLWH on AART regimens that are active against HBV and inappropriate ART discontinuation may lead to a flare of HBV infection [13]. The aim of this study was thus to determinate the prevalence of HBV-HIV co-infection among PLWH, the risk factors associated with co-infection, and the association between co-infection and the choice of ART and treatment outcomes among HIV patients in a busy HIV clinic in a referral hospital in Northern Uganda.

Materials and methods

This cross-sectional study involved 1000 respondents aged 13 years or older, selected by systematic random sampling of patients attending the HIV clinic of Saint Mary’s Hospital Lacor, the largest hospital in Northern Uganda, from April to June 2016. The AIDS outpatient clinic had cumulatively cared for 14,962 patients by 2015, of whom 6,217 were actively receiving ART [22]. Inclusion age was chosen because those below 13 years had received HBV vaccination that was introduced into Uganda’s vaccination programme in 2002 [23]. Patients requiring emergency care at the time of recruitment were excluded from the study.

Regardless of treatment status, participation in the study was offered to one in every 2–4 patients (depending on the number of visits expected each day) in their order of arrival at the outpatient clinic with the aim of randomly and consecutively recruiting 25 patients a day in order to reach a total of 1000 cases after a pre-fixed series of 40 days, starting on 12th April 2016. Sample size was calculated using the Leslie-Kish formula, a confidence level of 95%, a precision of 1.9%, and the estimated prevalence of HBV infection in Uganda (10.3%) [6]. Written informed consent was obtained from all participants. Respondents aged 13–17 years provided assent in addition to consent by their legal guardians.

A questionnaire was administered by nurses trained to collect participants’ age, sex, ethnic group, religion, residence, childhood (type of delivery and HBV status of mother, type of habitation, the number of siblings and cohabiting people, and the presence of HBV-infected persons). Other variables included displacement in an internal refugee camp during war, level of education, type of employment, history of military service and sexual history, circumcision, scarification, the use of injected drugs, blood transfusions, marital status and type of marriage (monogamous/polygamous), number of children, and HIV treatment adherence. Respondents’ clinical data (date of HIV diagnosis, ART and AIDS status, CD4+ T cell counts at the time of HIV diagnosis, start of ART, and after 6, 12 and 18 months of therapy, WHO clinical stage) were obtained from the patient records.

Upon enrolment, all participants were tested for HBsAg (One-Step HBsAg Test, InTec Products Inc., Xiamen, China; sensitivity 98.89%, specificity 98.87%), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (LiquiUV System reagent for HumaStar600, Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) in accordance with the standard procedures of the manufacturer adapted at St. Mary’s Hospital Lacor.

We also measured CD4+ T cell counts if not tested during the previous six months, and assessed HIV viral load if not tested during the previous twelve months.

The study received ethical approval from the Lacor Hospital Institutional Research and Ethics Committee and the Uganda National Council for Science and Technology (number HS 2034) in accordance with Ugandan regulations. Patient information was kept confidential and was accessible only by the study investigators.

Statistical analysis

Data were entered in an Excel spreadsheet, and analysed using SAS 9.4 and XLSTAT software. Wald’s test was used to calculate the population prevalence of HBV, and bivariate analysis was used to identify risk factors for HBV infection using the χ2 test or Fisher’s exact test for categorial variables, and the Mann-Whitney test for the quantitative variables. The nominal level of significance was set at α = 0.05. Factors associated with HBsAg positivity were analysed using univariate and multivariate logistic regression, and a general linear model was used to assess CD4+ T cell response to ART.

Results

The study recruited 1000 PLWH over 44 days at the outpatient clinic (a mean of 22 subjects per day); 15 subjects were excluded because r HBsAg was not tested after completing the survey, leaving a total of 985 subjects. 35 subjects not receiving ART would not affect the results of the statistical analysis, these were also excluded, and the final analysis was based on the 950 receiving ART.

The study population was majorly females (736, 77%), Catholic (74.3%), and Acholi ethnic group (92%) as shown in Table 1. Median age was 42 years (IQR: 35.0–49.9). 46% declared illiteracy, and only 17% had had more than a primary school education. Only 17.3% reported having formal or informal employment, the most frequent being “teacher” and “business”, whereas 81.2% worked as peasant farmers or were unemployed. A history of military service was infrequent (5.5%), while 49.9% of the study participants had stayed in an internally displaced persons (IDP) camp during the insurgency of the Lord’s Resistance Army (LRA).

Table 1

Socio-economical and behavioural characteristics of the 950 respondents.

	TOTAL		HBsAg positive		HBsAg negative		P value*
	N = 950		n = 75		n = 875
Age in years, median (IQR)							0.368
	42.8	(35.0–49.9)	42.4	(33.7–48.2)	42.8	(35.4–50.0)
Gender, N (%)							0.020
Male	214	(22.53)	25	(33.33)	189	(21.60)
Female	736	(77.47)	50	(66.67)	686	(78.40)
Religion, N (%)							0.139
Catholic	706	(74.32)	56	(74.67)	650	(74.29)
Anglican/Protestant	142	(14.95)	14	(18.67)	128	(14.63)
Pentecostal	45	(4.74)	5	(6.67)	40	(4.57)
Muslim	12	(1.26)	0	(0.00)	12	(1.37)
Other	45	(4.74)	0	(0.00)	45	(5.14)
Tribe, N (%)							0.134
Acholi	875	(92.11)	66	(88.00)	809	(92.46)
Lango	35	(3.68)	6	(8.00)	29	(3.31)
Other	40	(4.21)	3	(4.00)	37	(4.23)
Level of education, N (%)							0.475
No formal education	438	(46.11)	28	(37.33)	410	(46.86)
Completed primary school	334	(35.16)	30	(40.00)	304	(34.74)
Completed senior school	123	(12.95)	13	(17.33)	110	(12.57)
Qualification afther senior six	38	(4.00)	3	(4.00)	35	(4.00)
ND+	17	(1.79)	1	(1.33)	16	(1.83)
Employment, N (%)							0.375
Unemployed	267	(28.11)	23	(30.67)	244	(27.89)
Peasant farmer	410	(43.16)	26	(34.67)	384	(43.89)
Informal employment	152	(16.00)	17	(22.67)	135	(15.43)
Formal employment	108	(11.37)	8	(10.67)	100	(11.43)
ND	13	(1.37)	1	(1.33)	12	(1.37)
Soldier, N (%)
Yes	52	(5.47)	3	(4.00)	49	(5.60)	0.559
No	898	(94.53)	72	(96.00)	826	(94.40)
Type of marriage, N (%)							0.144
Polygamous	429	(45.16)	27	(36.00)	402	(45.94)
Monogamous	429	(45.16)	42	(56.00)	387	(44.23)
Never married / ND	92	(9.68)	6	(8.00)	86	(9.83)
Number of lifetime sex partners, N (%)							0.331
≤ 5	843	(88.74)	64	(85.33)	779	(89.03)
> 5	107	(11.26)	11	(14.67)	96	(10.97)
Number of children, N (%)							0.893
0	77	(8.11)	7	(9.33)	70	(8.00)
1–4	514	(54.11)	41	(54.67)	473	(54.06)
≥ 5	359	(37.79)	27	(36.00)	332	(37.94)
Scarification, N (%)							0.804
Yes	317	(33.37)	26	(34.67)	291	(33.26)
No	633	(66.63)	49	(65.33)	584	(66.74)
Circumcision, N (%)							0.066
Yes	20	(2.11)	4	(5.33)	16	(1.83)
No	930	(97.89)	71	(94.67)	859	(98.17)
Blood transfusion, N (%)							0.475
Yes	113	(11.89)	7	(9.33)	106	(12.11)
No	837	(88.11)	68	(90.67)	769	(87.89)
Lived in a refugee camp, N (%)							0.039
Yes	474	(49.89)	46	(61.33)	428	(48.91)
No	476	(50.11)	29	(38.67)	447	(51.09)
People in the same house/hut during childhood, N (%)							0.477
0–1	27	(2.84)	4	(5.33)	23	(2.63)
2–5	405	(42.63)	32	(42.67)	373	(42.63)
6–9	354	(37.26)	25	(33.33)	329	(37.60)
> 9	164	(17.26)	14	(18.67)	150	(17.14)
Type of delivery, N (%)							0.059
natural childbirth	875	(92.11)	64	(85.33)	811	(92.69)
caesarean section	21	(2.21)	3	(4.00)	18	(2.06)
ND	54	(5.68)	8	(10.67)	46	(5.26)
HBV-infected person in the house/compound where the respondent spent his/her childhood, N (%)							0.002
Yes	55	(5.79)	12	(16.00)	43	(4.91)
No	698	(73.47)	49	(65.33)	649	(74.17)
ND	197	(20.74)	14	(18.67)	183	(20.91)
Maternal HBV positivity status, N (%)							0.610
Yes	7	(0.74)	1	(1.33)	6	(0.69)
No	635	(66.84)	50	(66.67)	585	(66.86)
ND	308	(32.42)	24	(32.00)	284	(32.46)

+ND: no answer/don’t known / not defined.

* p-values are for χ2 or Fisher’s exact test and Mann-Whitney test.

Most participants were currently or previously married; only 9.6% had “never married”. Half of all marriages were monogamous, and half were polygamous. 92% of the participants reported having children (five or more in 37.79% of cases). Multiple sexual partners and traditional scarification for therapeutic purposes were reported by 11.3% and 33.4% of the respondents respectively. Only 2.1% of the participants were circumcised, and 11.9% reported receiving a blood transfusion (Table 1).

Almost all respondents said that they were born vaginally, had at least one sibling, and grew up sharing their hut/house with at least two other people. When asked whether anyone was infected with HBV in the compound in which they spent their childhood, 5.8% answered affirmatively and 73.4% answered negatively. Only 0.7% answered that their mother was positive for HBV (Table 1).

Median time from ART initiation to study enrolment was 71.5 months (IQR: 40.6–108.8), and almost all recruited participants were receiving ART (950/985); 67.2% had an undetectable HIV viral load, below 20 copies/ml. Median CD4+ T cell count increased from 240 cells/μL (IQR: 144–325 cells/μL) at the time of starting ART to 521 (IQR:380–695 cells/μL) at most recent available test. Median AST and ALT levels were 32 U/L (IQR:26–39) and 24 U/L (IQR:19–32) respectively (Table 2). Most respondents were on tenofovir (TDF)-containing regimens. 87.4% of the respondents denied missing any doses of therapy.

Table 2

Clinical, virologic, immunological and therapeutic characteristics of the 950 respondents undergoing antiretroviral therapy (ART) by HBV status.

	TOTAL		HBsAg+		HBsAg-		P value*
	N = 950		n = 75		n = 875
WHO stage at ART initiation, N (%)							0.833
1	254	(26.74)	19	(25.33)	235	(26.86)
2	332	(34.95)	25	(33.33)	307	(35.09)
3	252	(26.53)	21	(28.00)	231	(26.40)
4	39	(4.11)	2	(2.67)	37	(4.23)
ND	73	(7.68)	8	(10.67)	65	(7.43)
Current AST (U/L)							0.057
Median (IQR)	32	(26–39)	34	(28–43)	32	(26–39)
Current ALT (U/L)							0.140
Median (IQR)	24	(19–32)	26	(20–36)	24	(19–32)
Current HIV viral load undetectable, N (%)							0.726
Yes	639	(67.26)	52	(69.33)	587	(67.09)
No	73	(7.68)	4	(5.33)	69	(7.89)
ND	238	(25.05)	19	(25.33)	219	(25.03)
CD4+ T cell count at ART initiation							0.295
Median (IQR)	240	(144–325)	249	(175–336)	239	(142–324)
CD4+ T cell count at 6 months of ART
Median (IQR)	363	(243–519)	331	(240–429)	368	(243–520)	0.422
CD4+ T cell count at 12 months of ART
Median (IQR)	391	(277–534)	370	(295–518)	395	(274–538)	0.742
CD4+ T cell count at 24 months of ART
Median (IQR)	410	(298–577)	355	(288–478)	421	(300–580)	0.143
Current CD4+ T cell count							0.025
Median (IQR)	521	(380–695)	485	(323–593)	524	(385–705)
Change in CD4+ T cell count (current-initial)							0.012
Median (IQR)	274	(135–441)	236	(87–341)	280	(144–448)
Time of ART initiation, N (%)							0.388
2002–2013	782	(82.32)	59	(78.67)	723	(82.63)
2014–2016	168	(17.68)	16	(21.33)	152	(17.37)
Current ART regimen, N (%)							0.307
3TC+TDF+PI/NNRTI+	825	(86.84)	68	(90.67)	757	(86.51)
Other++	125	(13.16)	7	(9.33)	118	(13.49)
Months of antiretroviral therapy							0.129
Median (IQR)	71.5	(40.6–108.8)	61.8	(36.4–99.3)	72.1	(41.3–109.7)

ND: no answer/don’t known / not defined. IQR: interquartile range. AST/ALT: aspartate aminotransferase/alanine aminotransferase.

NNRTI: Non nucleoside Reverse Transcriptase inhibitor.

+3TC: lamivudine, TDF: Tenofovir, PIù: Protease inhibitor.

* p-values are for χ2 or Fisher’s exact test and Mann-Whitney test.

++ The other regimens have lamivudine, which without TDF was not considered as optimally active against HBV.

Seventy-five of the ART-treated patients were HBsAg positive, a prevalence of 7.9% (95% CI:6.2–9.6%). Table 3 shows the predictors of HBsAg positivity. Prevalence of HBsAg was significantly higher among males (11.7% vs 6.8%; P = 0.020), but was not significantly influenced by age, ethnicity or religion. It was also significantly higher among the patients who reported growing up in the same compound as an HBV-positive person (P = 0.002), and among those who had been interned in an IDP camp (P = 0.039). Additional gender-stratified analysis did not significantly change the results.

Table 3

Predictors of HBsAg positivity in the population of 950 PWH receiving ART (logistic univariate and multivariate analysis).

Univariate					Multivariate
	OR	OR lower limit (95%)	OR upper limit (95%)	p-value	OR	OR lower limit (95%)	OR upper limit (95%)	p-value
Gender
Female	1				1
Male	1,815	1,094	3,011	0,021	1,574	0,867	2,857	0,136
Religion
Catholic	1				1
Anglican/Protestant	1,299	0,707	2,389	0,400	1,096	0,541	2,219	0,800
Pentecostal	1,564	0,610	4,005	0,352	2,121	0,787	5,719	0,137
Muslim	0,461	0,024	8,840	0,607	0,523	0,030	9,272	0,659
Other	0,127	0,007	2,146	0,152	0,122	0,008	1,954	0,137
Tribe
Acholi	1				1
Lango	2,536	1,017	6,327	0,046	2,317	0,829	6,480	0,109
Other	0,994	0,298	3,310	0,992	1,170	0,303	4,515	0,820
Type of marriage
Monogamous	1				1
Polygamous	0,619	0,374	1,024	0,062	0,852	0,488	1,487	0,573
never married/ND	0,643	0,265	1,560	0,329	1,168	0,475	2,872	0,734
Circumcision
No	1				1
Yes	3,025	0,985	9,289	0,053	1,318	0,292	5,946	0,719
Refugee camp
No	1				1
yes	1,657	1,022	2,686	0,041	1,760	1,039	2,982	0,036
Type of delivery
natural childbirth	1				1
caesarean section	2,112	0,606	7,360	0,241	2,076	0,532	8,108	0,293
ND	2,204	0,997	4,869	0,051	1,552	0,602	4,004	0,363
HBV in the compound
no	1				1
yes	3,696	1,831	7,463	<0,001	3,309	1,495	7,324	0,003
ND	1,013	0,547	1,876	0,967	0,786	0,399	1,548	0,486
Current ALT (x 1 U/L)
	1,006	0,997	1,015	0,221	1,003	0,993	1,012	0,609
ΔCD4+ T cell (x 100 cells/mm3)
	0,910	0,840	0,986	0,022	0,884	0,791	0,988	0,030
Months of antiretroviral therapy (x 1 month)
	0,995	0,989	1,002	0,140	0,997	0,990	1,004	0,411

ND: NO Answer/Don’t Know/not defined. OR: Odds Ratio. ALT: Alanine aminotransferase.

All variables with a P-value of < 0.2 at univariate analysis were included in a multivariate analysis (Table 3), under which sex lost its statistical significance. Having lived in an IDP camp (adjusted odds ratio (AOR) 1.76, 95% CI 1.03–2.98; P = 0.036) or in close contact with a HBV-infected person during childhood (AOR 3.30, 95% CI 1.49–7.32; P = 0.003) however proved to be independent risk factors for HBsAg positivity. A Lower CD4+ T cell count was associated with HBsAg positivity (AOR 0.88, 95% CI: 0.79–0.98; P = 0.030). Rise in CD4+ T cells count in HIV-HBV coinfected patients was significantly less, compared to their HBV negative counterparts(Fig 1 and Table 3), and was independently associated with a higher pre-therapy CD4+ T (β = -0.48, 95% CI -0.54 to -0.42; P<0.0001), male sex (β = -0.15, 95% CI -0.21 to -0.09; P<0,0001) and HBsAg positivity (β = -0.06, 95% CI -0.12 to -0.008; P = 0.027), whereas the response was better the longer the therapy lasted (β = 0.08, 95% CI 0.02–0.14; P = 0.004) (Table 4).

Fig 1

Box plot showing the change in CD4+ T cell count from pre-ART initiation level to the most recent in HBsAg positive and negative patients.

Table 4

Predictors of CD4+ T cells response to ART therapy in the population of 950 PLWH (general linear model).

	Beta	Standard error	Pr > |t|	Lower CI (95%)	Upper CI (95%)
CD4+ T cells-start of therapy	-0,484	0,030	< 0,0001	-0,543	-0,425
Months of antiretroviral therapy	0,087	0,030	0,004	0,028	0,146
Stayed in refugee camp (yes vs. no)	-0,017	0,030	0,575	-0,075	0,041
Gender (m vs. f)	-0,154	0,030	< 0,0001	-0,213	-0,095
HBV in the compound (yes vs. no)	0,003	0,030	0,919	-0,055	0,061
HBV in the compound (ND vs. no)	-0,002	0,030	0,935	-0,060	0,056
Circumcision (yes vs. no)	-0,005	0,030	0,864	-0,064	0,053
HBsAg (yes vs. no)	-0,065	0,029	0,027	-0,123	-0,008
Tenofovir in start therapy (yes vs. no)	-0,047	0,030	0,114	-0,106	0,011

ND: No answer/don’t know/not defined.

Although their HBV status was unknown before this study was carried out, a higher percentage of HBsAg-positive than HBsAg-negative patients were receiving TDF (P = 0.032), and only four HBsAg-positive patients were being treated with zidovudine+lamivudine (AZT/3TC). TDF was included in the first-line ART regimen of 66 of the 68 HBsAg-positive patients currently being treated with it (Table 2).

There was no correlation between HBV coinfection and the other clinical, viral or biochemical variables analysed.

Discussion

This study found that HBV infection prevalence among HIV infected patients in a busy hospital clinic in Northern Uganda was 7.9%, and identified three independent factors associated with co-infection: living in an IDP camp between 1989 and 2006 [9, 24], cohabitation with HBV-infected persons during childhood, and a poorer CD4+ T cell count response to ART that underscores the negative impact of chronic HBV infection on HIV immunological response [20].

Although consistent with the results of some recent national and regional studies carried out in Uganda [8, 25], this prevalence was lower than the 17.9% reported in the general population in the same area in 2010 [11], the 10.8% prevalence found among pregnant HIV positive mothers in the same hospital in 2014 [26], and the 21.3% prevalence reported earlier across the whole Northern Uganda [6]. However, coinfection prevalence was similar to the prevalence of HBV co-infection in Europe [18, 27–29], North America [12, 30], and even in sub-Saharan Africa [31], where the reported ranges are wider than in Western countries and can reach 36% [5]. In countries where HBV is highly endemic, most cases of infection are attributable to vertical or horizontal transmission during early childhood [23, 32–34], although the findings of a recent study challenge the predominant role of vertical transmission in the area of Gulu [35]. This lower than expected prevalence raises the question as to whether there is a true decline in HBV prevalence in the region.

Our interviewers reported that many study participants had not heard of HBV until recently, thus most respondents did not know their maternal HBV infection status. However, it was found that being aware of the presence of HBV-infected subjects in the compound where a participant was brought up was independently associated with a higher risk of being HBsAg positive (P = 0.002).

Interestingly, having lived in an IDP camp was also an independent risk factor associated with HBV infection (P = 0.039). It is possible that life in these camps, which was characterised by greater crowding and higher risk sexual behaviours than regular village life, increased the likelihood of horizontal HBV transmission in our patients whose median age at the time ranged from 15 to 32 years. According to official data, more than 90% of Northern Uganda residents had lived in IDP camps by 2005 [10], but only 50% of the study participants reported it. This was possibly because those who had not been permanent residents (people who only spent nights in the camps and moved back to their own homes during the daytime) may have answered the question negatively.

The lower prevalence of HBV than that found in the general population of the same area deserves some comments. First of all, 77% of our study were women because 66% of the patients attending the AIDS clinic of St. Mary’s Hospital are actually women [22]. This disproportion can be attributed to the practice of HIV testing during pregnancy [36] and the poor health-seeking behaviour of males [22]. In line with the findings of other African and European studies [5, 8, 27, 34, 37], the prevalence of HBV in our cohort was higher among men (11.7% vs 6.8%), but their under-representation may have lowered the actual overall prevalence. Secondly, our sampling procedures were performed in the AIDS clinic, and our study population consisted of regularly attending outpatients in good clinical condition with a high rate of adherence to ART (87.3%), which they had been receiving for a median of six years. Other studies of PLWH outpatients have reported comparable prevalence rates ranging from 6.7% to 11.5% [25, 26, 35] and, assuming that HIV/HBV co-infected patients experience a faster clinical evolution than other PLWH, it is possible that a disproportional number of co-infected patients were not enrolled [14, 27, 29]. Third, it cannot be excluded that mortality among the co-infected may have been higher than in the PLWH population as a whole, thus leading to a lower prevalence of HBV among the survivors. Nevertheless, the significant drop in prevalence noted in this and recent studies among different populations [8, 25] from values seen six to 12 years prior [6, 11] may signal the dawn of a decline in HBV prevalence in Northern Uganda.

The incidence of new chronic HBV infections is reduced in patients receiving lamivudine- and/or tenofovir-containing regimens and there is also a slim possibility of HBsAg seroreversion [38]. Despite this, the factors favouring horizontal transmission of HBV in our study population which is probably more relevant than vertical transmission suggest that consideration should be given to extending vaccine prophylaxis to HIV patients at risk.

The sensitivity and specificity of HBsAg rapid tests have recently been evaluated in a systematic review [31], which concluded that some perform better in the presence of high HBV viremia levels. As a potential limitation, the test we used was not included in this review and so we do not know whether it shares the same limitation. However, as the vast majority of our HBsAg-positive patients had long been treated with tenofovir, it is likely that most of our study population had low viremia levels. If the sensitivity of our test depends on HBV viremia levels which we did not perform, this may be a limitation of the study. Of note also, the Ugandan comparator studies used similar HBsAg tests.

Similar to other studies carried out in high [18, 20]- and low-income countries [39], we found that HBV-HIV co-infection was associated with a worse immunological response to ART. Moreover, shorter-lasting ART, a lower baseline CD4+ T cell count and male sex also had independently negative effects on immunological outcomes. Assuming poor health-seeking behaviour among men and a worse natural history of co-infection, all these factors seem to be strictly related to each other.

Finally, although they were not involved in HBV transmission in the study population, polygamy and blood transfusion were significantly frequent: the former because it is customary for Acholi men to have more than one official wife, the latter because P. falciparum malaria is widespread in the area with potential for consequent severe anaemia [22].

Our finding that having lived in an IDP camp was associated with higher HBV prevalence, (something that is conceivably common to all IDP camps in areas that are highly endemic for HBV), as well as the worse CD4+ T cell response to ART in HBV-HIV coinfected persons supports the need to extend preventive interventions, particularly HBV screening and vaccination, to people at risk in this kind of emergency setting or in high HBV prevalence areas, coupled with prompt initiation of appropriate treatment for those positive. We also recommend further research to confirm the apparent drop of HBV prevalence in the Northern region of Uganda.

Data_HBV_HIV_Uganda.

(XLSX)

Click here for additional data file.

Questionnaire.

(PDF)

Click here for additional data file.

Translated questionnaire.

(PDF)

Click here for additional data file.

6 Aug 2020

PONE-D-20-20265

Hepatitis B and HIV coinfection in Northern Uganda: is a decline in HBV prevalence on the horizon?

PLOS ONE

Dear Dr. Ochola,

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3. We note that part of your methods state that patients were selected starting on 23 May 2016, whereas earlier in the methods April - June 2016 is cited. Please clarify the date your study began.

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6. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

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Additional Editor Comments (if provided):

This is a cross-sectional study of HBV infection among persons living with HIV in Uganda.  Given the burden of viral hepatitis in sub-Saharan Africa, the need for such studies is high.

The population size is good at >950 individuals.  The overall prevalence of 7.9% is what would be expected for a country in sub-Saharan Africa.  However, this manuscript would benefit from careful review by a native English speaker and/or a professional editing service.

The authors should clarify what population reference 10 was conducted in . . . was this the general population or persons living with HIV?

Was the data collection questionnaire self-administered or conducted by a researcher or clinician?

How was HIV treatment adherence reported and confirmed?

The authors should comment on how many individuals receiving ART were receiving HBV-active drugs as part of their ART regimen.  These data on specific ART regimens is confusing.  Tenofovir is mentioned but what about 3TC?  Are they always given together or could some individuals receive tenofovir only or 3TC only?

It appears that HBV DNA testing was not conducted.  This and the lack of information on HBV genotypes should be mentioned explicitly as limitations in the discussion.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study determined the prevalence, correlates of the risk of HBV infection, and the effect of co-infection on the outcomes of ART among people living with HIV in Northern Uganda. The study enrolled 1000 participants and screened for HBsAg using rapid ELISA. An HIV/HBV coinfection prevalence of 7.9% was reported in participants who were on ART. Majority of the participants were on a Tenofovir containing. The study filled an important gap in the field. I would like to commend the authors. The study was well conducted and the article well written. There are some minor changes and suggestions which might improve the study.

Major revision

1. The study speaks of a decline in HBV prevalence based on comparison with a previous population based study. However, there were differences in the studied populations. The population in the recent study was mostly on Tenofovir a potent anti-HBV drug for a median duration of 6 years. It is expected that the prevalence will be lower due to treatments effects as some patients might have lost the HBsAg. The population based 2010 survey might have included HIV negative participants who were not on HBV active treatment or people living with HIV but not on HAART. The ‘decline would have been better ascertained if it was compared to previous treatment experienced patients in the same population. Furthermore, the study admits to the possibility of the HBsAg underestimation due to the kit used, which was not reviewed for performances in low HBV levels. The prevalence reported was also similar to studies in similar populations elsewhere.

Minor revisions

Abstract

2. People living with HIV/AIDS (PLWHA) should be changed to ‘People living with HIV (PLWH)’ according to the NIAID HIV Language Guide (February 2020)

3.The study type and time of study enrolment is missing.

4. HbsAg under Patients and Method should be changed to HBsAg

5. Shored in results section should be corrected to shared

6. CD4+ cell should be corrected to CD4+ T cell here and elsewhere in the article.

Introduction

7. WHO should be written in full since it’s first mention.

8. There is a space missing between the sentence (page 3) ‘Furthermore, as Northern Uganda was the theatre of a civil war from 1989 to 2006’ and the reference.

9. HIV clients should be changed to people living with HIV (PLWH)

Materials and methods

10.Page 5, In the sentence A questionnaire was administered to collected information’, the word collected should be corrected to collect

11. Page 5, AST and ALT should be written in full at first mention.

12. Page 6: The authors mention that testing was done ‘in accordance with the standard procedures of St. Mary’s Hospital Lacor’. Are the procedures different from the manufacture’s protocols? If they are then the differences should be noted for ease of reproducibility since the hospital’s procedures were not referenced.

13. In the sentence ‘We also made CD4+ T cell counts’, made should be changed to measured

Results

14. The authors mention that the 35 subjects excluded were not going to affect results but did not qualify the statement as to why/how they were not going to affect results.

15. Include explanations of all abbreviations below tables.

16. Page 10. The authors states that ‘All of the variables with a P-value of >0.2 at univariate analysis were included in a multivariate analysis, but table 3 includes variables with p values which were at univariate analysis < 0.2

17. Table 4 is not uniform. Some variable are written in all capital letters while others are written in sentence case.

Discussion

The discussion is well written with sound conclusions.

Reviewer #2: This manuscript reports on the findings of a study aimed at exploring a potential shift in the prevalence of chronic hepatitis B virus (HBV) infection (based on the prevalence of HBsAg) among people living with HIV and AIDS (PLWHA) in Northern Uganda. Given intensified global efforts towards elimination of viral hepatitis by 2030, the findings of this study could help inform public health strategies particularly in endemic regions such as sub-Saharan Africa. I have the following comments on the manuscript that need addressing;

Major revisions

In the Discussion section on page 14, the authors state the following;

“This leaves a new question to confirm: is there a true decline in HBV prevalence in the region?”.

To better address the hypothesis of the study, could the authors clearly comment on how the following could have impacted on the prevalence of HBsAg found within the study population:

•The study made use of a health facility-based population that may have different health-seeking behaviour from PLWHA within the general population. In addition, the fact that 86.8% of the study population were on long-term HBV active regimens (3TC and TDF) could reduce the prevalence of HBsAg.

•It is well established that the burden of occult HBV infection (OBI) is higher among PLWHA than the general population. Given that the prevalence of OBI was not assessed as part of this study, could the burden of HBV infection have been underestimated – could the lack of testing for anti-HBc and HBV DNA been a limitation to fully understanding the proportion of PLWHA who had not had been infected with HBV?

Minor revisions

•Abstract

o“…selected to undergo a rapid hepatitis B surface antigen (HbsAg) after administering a questionnaire.” Insert the word “test” after “HBsAg” for better clarity.

o“…and having shored housing with HBV-infected people…” Do the authors mean “shared”?

•Introduction

oPage 3; "About 5-15% of persons living with HIV worldwide have hepatitis B virus (HBV) infection". The current Global Hepatitis Report (2017) compiled by the WHO estimates that the global prevalence of HBV infection in HIV-infected persons is 7.4%. I would suggest this as a more appropriate and up-to-date reference.

oPage 3; “…and a high prevalence in Chad, Cameron, and western Africa (≥8%)…” Do the authors mean “Cameroon”?

oPage 4; “…co-infected subjects more rapidly develop liver fibrosis…and respond less to HBV vaccine.” Could the authors elaborate on this for better clarity? Given that the hepatitis B vaccine is a preventative and not a therapeutic vaccine, it would not be administered to those who are already infected.

•Results

oPage 9; “…almost all the recruited participants were receiving ART (950/985)…” In the methods sections, the authors clearly indicate that all 35 participants who were yet to initiate ART had been excluded from analysis.

oIn Table 2, does the line item “Months of therapy” refer to the number of months participants have been on ART or some other form of therapy?

oPage 10; “…and this was independently associated a higher CD4+ T cell count at the time…” insert the word “with” after “associated”.

•Discussion

oPage 15; “…crowding and promiscuity than that associated with village life…” I would suggest that the authors replace the word "promiscuity" with "high risk sexual behaviour" if this is indeed what they are referring to.

oPage 16; “Finally, although they were not involved in HBV transmission…customary for Acholi men to have more than one official wife, the latter because P. falciparum malaria is widespread in the area.” If these findings have no bearing on the burden of HBsAg or the risk of HBV transmission within the population, I would suggest that the authors provide some clarity as to why it has been highlighted in the discussion section.

**********

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Reviewer #1: No

Reviewer #2: Yes: Edina Amponsah-Dacosta

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30 Sep 2020

We thank the reviewers for the very constructive comments to improve our paper.

We have attached all the responses in the "Response to reviewers" file. Each issue is specifically responded to, and reference is made to enable such changes to be located on the tracked and unmarked manuscripts. Here below we only list the issues and responses, but the table referred above gives a more organised response.

Thank you again

Dr. Emmanuel Ochola (on behalf of the authors)

EDITOR'S COMMENTS

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming

Response (RS):

The file and supporting information naming as well as style have been modified to fit Plos One requirements.

2. Thank you for stating in the text of your manuscript "Written informed consent was obtained from all the participants or, in the case of those aged 13-17 years, their legal guardians". Please also add this information to your ethics statement in the online submission form

RS

This information has been added to the ethics statement in the online submission form.

3. We note that part of your methods state that patients were selected starting on 23 May 2016, whereas earlier in the methods April - June 2016 is cited. Please clarify the date your study began

RS

We apologize for this mistake. The actual dates of interview and HBsAg sampling were from 12th April to June 17th. The mistake in dates have now been corrected.

4. Please provide more details on the limitations of your study methods

RS

The limitations of the HBsAg underestimating the prevalence is not unique to this, but also applies to similar prevalence studies in Uganda. The HIV specific population and treatment on tenofovir potentially lowering prevalence have been discussed in a paragraph under discussion

5. We note that you state "CD4+ cell counts were significantly lower in the HBV patients (p=0.025)" however Table 2 shows the opposite. Please revise your table if, for example, values were swapped accidentally

RS

Thanks for noting this. However, the only time that CD4 was higher for the HBV positive than the HBsAg negative was at baseline. In all the other times after ART initiation, the CD4_+ cell count was higher for the HBsAg positive. The p value of 0.025 was noting that the rise in CD4+ count from baseline to current level is higher for HBsAg negative participants. No change has been made

6. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information

RS

The two versions of the questionnaire have been added as supporting information

7. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the

minimal data set be made fully available. For more information about our data policy, please see

http://journals.plos.org/plosone/s/data-availability

RS

We thank the Editor for these comments.

We had earlier submitted the data as additional supporting information, in line with Plos recommendations. In this resubmission, we attach the final combined dataset as supporting information. However, we have not deposited it in any other specific online location.

8. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs,

DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories,

please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized

RS

9. Specific identifying materials have been removed and dataset attached

The population size is good at >950 individuals. The overall prevalence of 7.9% is what would be expected for a country in sub-Saharan Africa. However, this manuscript would benefit from careful review by a native English speaker and/or a professional editing service

RS

We thank you for the kind comment about sample size. Our point in this paper is that for the specific higher burden Northern Ugandan region. The described prevalence is lower than previous findings.

Proof reading (not professional editing) has been done leading to changes in sentence constructions, flow and edits in the document. We have duly acknowledged this service

10. The authors should clarify what population reference 10 was conducted in . . . was this the general population or persons living with HIV?

RS

The referenced study was done in the general population, with and without HIV. A statement “with no documentation of HIV status” has been added

11.Was the data collection questionnaire self-administered or conducted by a researcher or clinician?

RS: The questionnaires were administered by nurses trained as research assistants. All of them had experience in HIV care. A statement to that effect has been added

12. How was HIV treatment adherence reported and confirmed?

RS: Adherence was measured using a composite of self-reporting and pill count, or recorded as assessed and documented by the primary health provider

13. The authors should comment on how many individuals receiving ART were receiving HBV-active drugs as part of their ART regimen. These data on specific ART regimens is confusing. Tenofovir is mentioned but what about 3TC? Are they always given together or could some individuals receive tenofovir only or 3TC only?

RS: The Primary backbone regimen in use in Uganda has been Tenofovir/Lamivudine fixed dose combination, usually in addition to EFV or NVP. All the patients an ART the clinic actually take Lamivudine in combination with other drugs. We did not consider Lamivudine combined with Abacavir or Zidovudine to be an optimal treatment- as guided by the Uganda HIV treatment guidelines. 86.84% percent were on Lamivudine/Tenofovir Diopropyl Fumerate and 13.16% were on Lamivudine combined with zidovudine or Abacavir, as part of their Antiretroviral treatment.

Finally, we did not mention treatment with lamivudine-only because of the low rate of HBsAg seroconversion seen with this regimen, which may not change the results of our study

14. It appears that HBV DNA testing was not conducted. This and the lack of information on HBV genotypes should be mentioned explicitly as limitations in the discussion

RS: Given the prohibitive cost of HBV DNA testing and the high number of positive respondents, we were unable to provide testing. We have added a statement about the potential better performance of HBsAg in high HBV viraemia, as a limitation under Discussion section

REVIEWER 1

The study speaks of a decline in HBV prevalence based on comparison with a previous population based study. However, there were differences in the studied populations. The population in the recent study was mostly on Tenofovir a potent anti-HBV drug for a median duration of 6 years. It is expected hat the prevalence will be lower due to treatments effects as some patients might have lost the

HBsAg. The population based 2010 survey might have included HIV negative participants who were not on HBV active treatment or people living with HIV but not on HAART. The ‘decline would have been better ascertained if it was compared to previous treatment experienced patients in the same population. Furthermore, the study admits to the possibility of the HBsAg underestimation due to the

kit used, which was not reviewed for performances in low HBV levels. The prevalence reported was also similar to studies in similar populations elsewhere

RESPONSE (RS): The assertions about a decline in prevalence compares many previous studies to current ones, not just the 2010 study, as articulated under discussion, lines 225 to 229.

As reported in the “Discussion” paragraph, we are aware that it is not possible to compare a prevalence study conducted on general population with one conducted on an HIV-positive group. However, data about Northern Uganda are still few, so we had to use all the available sources.

We discussed why our population may have biases with the general population that explains this result (gender disproportion, we excluded all patients with critical conditions, we recruited potentially more adherent).

Although it is true that long term TDF therapy may lead to HBsAg seroconversion, as stated in 2017 EASL guidelines this is a really rare event (3% after 48-52 weeks of TDF treatment in HBeAg positive patients and 0% after 48-52 weeks of TDF treatment in HBeAg negative patients). The bias is possible but of minor entity. Thus the decline may not be negligible

2. People living with HIV/AIDS (PLWHA) should be changed to ‘People living with HIV (PLWH)

RS: 2. Thanks for the suggestion. This has been corrected in 10 instances and now appears as PLWH

3. Study type and time of enrolment is missing

RS: Study type: cross sectional study, and period (April to June) now included. This has been corrected, page 2 lines 30 and 33

4. HbsAg under Patients and Method should be changed to HBsAg

RS: Thanks for this. Four instances were corrected in text, and multiple instances in the tables.

5. Shored in results section should be corrected to shared

RS: Shored now appears as shared

6. CD4+ cell should be corrected to CD4+ T cell here and elsewhere in the article.

RS: Thanks for the suggestion. We have corrected multiple incidences in the text and tables

Introduction

7. WHO should be written in full since it’s first mention.

RS: Thanks, this has been corrected

8. There is a space missing between the sentence (page 3) Uganda was thetheatre of a civil war from 1989 to 2006’ and the reference.

RS: This space has now been included

9. HIV clients should be changed to people living with HIV (PLWH)

RS: Thanks for these, which have been corrected as shown under 2 above

Materials and methods

10. Page 5, In the sentence A questionnaire was administered to collected information’, the word collected should be corrected to collect

RS: We appreciate this comment. The word “collected” now appears a s “collect”

11. Page 5, AST and ALT should be written in full at first mention.

RS: Corrected as suggested

12. Page 6: The authors mention that testing was done ‘in accordance with the standard procedures of St. Mary’s Hospital Lacor’. Are the procedures different from the manufacture’s protocols? If they are then the differences should be noted for ease of reproducibility since the hospital’s procedures were not referenced.

RS: The Standard operating procedures do not vary. We have now added “in accordance to the standard operating procedures of the manufacturers adapted at St. Mary’s hospital Lacor

13. In the sentence ‘We also made CD4+ T cell counts’, made should be changed to measured

RS The word measured is now in place of made

Results

14. The authors mention that the 35 subjects excluded were not going to affect results but did not qualify the statement as to why/how they were not going to affect results

RS: Because of the sample size, the results of a statistical analysis conducted on 985 or 950 subjects did not gave significantly different results. Consequently, we decide to present in the paper the analysis on the 950 patients undergoing ART to have a more homogeneous sample to discuss

15. Include explanations of all abbreviations below tables.

RS: These have now been included where elsewhere missing.

16. Page 10. The authors states that ‘All of the variables with a P-value of >0.2 at univariate analysis were included in a multivariate analysis, but table 3 includes variables with p values which were at univariate analysis < 0.2

RS: Thanks for noting this mistake, which has now been corrected to p values <0.2%

17. Table 4 is not uniform. Some variable are written in all capital letters while others are written in sentence case.

RS: Table 4 now corrected to have uniform non capitalized letters

Discussion

The discussion is well written with sound conclusions.

RS: Thanks for the encouraging comment

REVIEWER 2

MAJOR REVISIONS

In the Discussion section on page 14, the authors state the following;

“This leaves a new question to confirm: is there a true decline in HBV prevalence in the region?”.

To better address the hypothesis of the study, could the authors clearly comment on how the following could have impacted on the prevalence of HBsAg found within the study population:

•The study made use of a health facility-based population that may have different health-seeking behaviour from PLWHA within the general population. In addition, the fact that 86.8% of the study population were on long-term HBV active regimens (3TC and TDF) could reduce the prevalence of HBsAg.

•It is well established that the burden of occult HBV infection (OBI) is higher among PLWHA than the general population. Given that the prevalence of OBI was not assessed as part of this study, could the

burden of HBV infection have been underestimated – could the lack of testing for anti-HBc and HBV DNA been a limitation to fully understanding the proportion of PLWHA who had not had been

infected with HBV?

RS:

Similar concerns have been answered under response to major revisions by Reviewer 1.

Most of the discussion focuses on the prevalence found.

The seeming decline in prevalence seen in this study is not an isolated finding. First, the population prevalence of HIV at the time was about 8.3%, and this is from the whole region.

Because of the low rate of HBsAg seroconversions anticipated with 3TC/TDF, our result should not be heavily biased by those therapy.

Of course the lack of data about HBV-DNA and HBcAb is a limitation, but

even when we anticipate OBI to reduce prevalence in HIV, the prevalence is actually lower than previous (Bwogi et al, Ochola et al), but higher than a similar study done around the same time in the same region (UPHIA report).A previous study conducted among women attending antenatal care (ANC) of the same area found HBV positive among 10.81% HIV positive pregnant women. Such leaves the question on decline as something to be confirmed.

Lack of HBV DNA testing has been noted as a limitation answered above under the last comment by the Editor, as well as to Reviewer 1.

MINOR REVISIONS

Abstract

1. “…selected to undergo a rapid hepatitis B surface antigen (HbsAg) after administering a questionnaire.” Insert the word “test” after “HBsAg” for better clarity.

RS: Thanks for the suggestion, “Test” has been inserted

2.“…and having shored housing with HBV-infected people…” Do the authors mean “shared”?

RS: The right word “shared” now appears in place of shored

Introduction

3.Page 3; "About 5-15% of persons living with HIV worldwide have hepatitis B virus (HBV) infection".

The current Global Hepatitis Report (2017) compiled by the WHO estimates that the global prevalence of HBV infection in HIV-infected persons is 7.4%. I would suggest this as a more appropriate and upto-date reference.

RS: We thank the reviewer for this suggestion, which has now been integrated in our references

4.Page 3; “…and a high prevalence in Chad, Cameron, and western Africa (≥8%)…” Do the authors mean “Cameroon”?

RS: Thanks. We meant Cameroon, now correctly written

5. Page 4; “…co-infected subjects more rapidly develop liver fibrosis…and respond less to HBV vaccine.” Could the authors elaborate on this for better clarity? Given that the hepatitis B vaccine is a preventative and not a therapeutic vaccine, it would not be administered to those who are already infected.

RS: What we describe is how PLWH respond to HBV infection or HBV vaccine. We agree that there would be no point in giving HBV vaccine to people already infected by HBV virus. In order to eliminate this misunderstanding, we modified the phrase to exclude vaccination

Results

6.Page 9; “…almost all the recruited participants were receiving ART (950/985)…” In the methods sections, the authors clearly indicate that all 35 participants who were yet to initiate ART had been excluded from analysis.

RS: The opening statement of results section does clarify on the exclusion of the 35 from analysis. They are not included in Table 2

7.In Table 2, does the line item “Months of therapy” refer to the number of months participants have been on ART or some other form of therapy?

RS: This has been corrected to read “months of antiretroviral therapy”, since it applies to duration on antiretroviral therapy

8..Page 10; “…and this was independently associated a higher CD4+ T cell count at the time…” insert the word “with” after “associated”.

RS: Thanks. “With” has now been included

Discussion

9.Page 15; “…crowding and promiscuity than that associated with village life…” I would suggest that the authors replace the word "promiscuity" with "high risk sexual behaviour" if this is indeed what they are referring to.

RS: Thanks for suggesting a more respectful word that has now replaced promiscuity

10. Page 16; “Finally, although they were not involved in HBV transmission…customary for Acholi men to have more than one official wife, the latter because P. falciparum malaria is widespread in the area.” If these findings have no bearing on the burden of HBsAg or the risk of HBV transmission within the population, I would suggest that the authors provide some clarity as to why it has been highlighted in the discussion section.

RS: Malaria is thought to predispose people to having severe anemia that might necessitate blood transfusion, a risk factor. These unstudied potential risk factors do exist in the community, hence the mention.

We have added that severe anemia is a potential consequence of P. falciparum infection. This is to support the reason for transfusion-based HBV risk

Submitted filename: Response to reviewers.docx

Click here for additional data file.

19 Oct 2020

PONE-D-20-20265R1

Hepatitis B and HIV coinfection in northern Uganda: is a decline in HBV prevalence on the horizon?

PLOS ONE

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Reviewer #2: Line 32; “Factors associated with HBsAg positivity were analysed univariate...” Please insert the word, “using” before “univariate”.

Line 165; “viral load, below 20 copies/μ.…” Please confirm the unit of measure for the viral load assessment.

Lines 206-207; “…HIV infected patients in area busy hospital clinic in Northern Uganda...” This sentence is unclear to me, kindly rephrase for better clarity.

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Reviewer #1: Yes: Motswedi Anderson

Reviewer #2: Yes: Edina Amponsah-Dacosta

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27 Oct 2020

Reviewer 2

1. Line 32; “Factors associated with HBsAg positivity were analysed univariate...” Please insert the word, “using” before “univariate”.

RESPONSE: We thank the reviewer for spotting this. We have now added the word “using” to make the statement understandable. This correction can be found on page 6 line 132 in both the tracked and clean documents.

2. Line 165; “viral load, below 20 copies/μ.…” Please confirm the unit of measure for the viral load assessment.

RESPONSE: The unit of viral load is copies per milliliters (ml). Therefore, ml has now replaced µ. Thank you very much. This is reflected in the tracked document on page 9 line 165 in the tracked document and on page 9 line 164 in the clean document.

3. Lines 206-207; “…HIV infected patients in area busy hospital clinic in Northern Uganda...” This sentence is unclear to me, kindly rephrase for better clarity

RESPONSE: Thank you for noting this. The word area was supposed to be only “a”. The phrase now reads, “This study found that HBV infection prevalence among HIV infected patients in a busy hospital clinic in Northern Uganda was 7.9%”. Page 14 line 206 in the tracked document and page 14 line 205 in the clean document.

Submitted filename: Response to reviewer comments.docx

Click here for additional data file.

30 Oct 2020

Hepatitis B and HIV coinfection in northern Uganda: is a decline in HBV prevalence on the horizon?

PONE-D-20-20265R2

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Kind regards,

Jason Blackard, PhD

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PLOS ONE

Additional Editor Comments (optional):

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Reviewers' comments:

None

6 Nov 2020

PONE-D-20-20265R2

Hepatitis B and HIV coinfection in northern Uganda: is a decline in HBV prevalence on the horizon?

Dear Dr. Ochola:

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