Highlighting a population-based re-emergence of Syphilis infection and assessing associated risk factors among pregnant women in Cameroon: Evidence from the 2009, 2012 and 2017 national sentinel surveillance surveys of HIV and syphilis.

BACKGROUND: Syphilis and HIV can be transmitted from pregnant women to their children and they remain a public health problem in Africa. Our study aimed to determine the trends of seroprevalence of HIV/syphilis co-infection and syphilis infection overtime through the national surveillance system in Cameroon and to explore associated risk factors.
METHODS: We conducted cross-sectional studies of HIV and syphilis, targeting each year 7000 first antenatal care (ANC-1) attendees at the same sites during the 2009, 2012 and 2017 sentinel surveillance surveys. Pregnant women were enrolled at their ANC-1, sociodemographic and clinical information were collected. HIV and Syphilis test were performed by serial algorithm as per the national guidelines. Trends were assessed for HIV, syphilis and HIV/syphilis by estimating seroprevalence from cross-sectional studies. Associated risk factors were explored using multinomial logistic regression with 4 outcomes: HIV/syphilis co-infection, HIV infection only, syphilis infection only and no infection.
RESULTS: Overall, 6 632, 6 521 and 6 859 pregnant women were enrolled in 2009, 2012 and 2017 respectively. In 2017, a total of 3 901 pregnant women enrolled were tested for syphilis. Almost half of them (47.9%) were living in urban area and were aged less than 25 years (44.7%). While HIV epidemic was on a decline (from 7.6% (95% CI: 6.99-8.28) in 2009 to 5.7% (95% CI: 4.93-6.4) in 2017), a huge significant increase of syphilis prevalence was observed (from 0.6% (95% CI:0.40-0.80) in 2009 to 5.7% (95% CI:4.93-6.40) in 2017). Pregnant women residing in rural areas were more likely to be infected with syphilis than those living in the urban area (aOR = 1.8 [95% CI: 1.3-2.4]). Unmarried pregnant women were three time more likely to be infected by HIV/Syphilis Co-infection than married, cohabiting, widow or divorced pregnant women (aOR = 2.8 [95% CI: 1.3-2.4]). Furthermore; living in Northern region was associated with a lower risk of being infected with HIV (aOR = 0.6 [95% CI: 0.5-0.9]) and Syphilis infection (aOR = 0.6 [95% CI: 0.4-0.9]).
CONCLUSION: The epidemiological dynamics of syphilis suggests a growing burden of syphilis infection in the general population of Cameroon. Our findings support the fact that while emphasizing strategies to fight HIV, huge efforts should also be made for strategies to prevent and fight syphilis infection especially among HIV positive women, in rural area, and southern regions.

Introduction

HIV remains a public health problem especially in Sub-Saharan African countries. In 2018, 37.9 (32.7–44.0) million people globally were living with HIV while about 32 (23.6–43.8) million people have died from AIDS-related illnesses since the start of the epidemic [1]. About 540 000 [470 000–590 000] of them were living in Cameroon with AIDS-related deaths of 18 000 [15 000–21 000] people in the same year [2].

In Cameroon, the recent Population-based HIV Impact Assessment (CAMPHIA) conducted in 2017 reported an HIV prevalence of 3.7% in the population aged 15–64 years [3]. This prevalence varied by region, ranging from 6.3 percent in the South region to 1.5 percent in the Far North region, and by gender: females been twice more infected than males (5% and 2.3% respectively) [3].

The introduction and continuous availability of highly effective antiretroviral therapy (ART) have significantly reduced the mortality among people living with HIV/AIDS (PLHIV) worldwide and have transformed AIDS from an inevitably fatal condition to a chronic and manageable disease in some settings [4].

Sexual Transmitted Infections (STIs) such as HIV and Syphilis remain a public health problem in Africa and worldwide [9]. Many sub-populations like blood donors, armed forces, Men who have Sex with Men (MSM) are affected by these diseases [5-10]. HIV and Syphilis are two of common pathogens to which many pregnant women in Sub-Saharan African Countries are exposed or infected [5-11]. They can be transmitted vertically during pregnancy to their children. Pregnant women diagnosed with early stages of syphilis are most at risk to transmit syphilis to their infants, 60% to 90% of pregnant women with untreated primary or secondary syphilis will transmit syphilis to their fetus as compared to less than 10% of women with late latent syphilis [12]. Access to screening during pregnancy has improved over the past decade in Sub-Saharan African Countries generally and in Cameroon particularly as HIV prevention programs have been scaled up. The Screening of such infections during antenatal care visit is important since many of these infections are asymptomatic [9].

Moreover, syphilis is an important risk factor in HIV transmission. Syphilis has been implicated in susceptibility to Human Immunodeficiency Virus (HIV) infection with an odds ratio of 8.5 for men and 3.3 for women [13]. If the infection remains undiagnosed, complications of late stage syphilis will develop in a portion of affected individuals [11].

Syphilis and HIV continue to be significant problems in Cameroon. Hence, identifying the risk factors and analyzing the trends would serve as a footprint to set-up evidence based interventions to reduce the burden of HIV and Syphilis among pregnant women and would benefit global efforts to eliminate both diseases in infants [14-16]. We therefore sought to describe the prevalence trends of syphilis infection and HIV/Syphilis co-infection among pregnant women using sentinel surveillance surveys data over a nine-year period and to investigate the associated risk factors.

Methods

Description of the surveys, study design and participants

To determine trends of syphilis prevalence over nine-year period, data from consecutive ANC cross sectional analytic surveys conducted in 2009, 2012 and 2017 were examined. As recommended by WHO for HIV sentinel surveillance survey [17], from each year, a cross sectional analytic study was conducted in 20 sentinels survey sites across the 10 Regions of Cameroon, which included 60 HIV surveillance health facilities (routine collection points). A sentinel site is a specific geographic area in the region. In the sentinel site, HIV surveillance health facilities (clinic and or hospital) were the data collection points. Each sentinel site had 3 data collection points. We have implemented a non-probabilistic approach which consisted of a systematic sampling method with two stages: (1) Selection of sentinel sites and surveillance health facilities (routine collection points), (2) Selection of pregnant women in each study sentinel site. The health facilities were chosen based on their capacity to provide both ANC and PMTCT services, their location (urban and rural settings in each region of the country), ANC attendance (capacity to enroll at least 300 pregnant ANC1 attendees during the study period of three months). At each site, pregnant women aged 15–49 years attending their first ANC were enrolled consecutively until the sample size was reached. The sample size for each region was determined as recommended by WHO guidelines [17] taking into considerations the observed prevalence of HIV and a desired precision of 95%. The minimum number of pregnant women at first ANC required to estimate the prevalence of HIV infection in that population with precision i = 0.03 and for a fixed risk α = 0.05 (Z = 1.96) is given by the following formula: where P = = observed prevalence

i = precision of the estimation

Zα/2 = Quantile for a risk α = 0,05

HIV and syphilis testing procedure

HIV testing

Plasma samples and sociodemographic data were collected as per the routine clinical practice per site. During the study period, HIV screening was proposed to every ANC-1 attendee according to the serial algorithm recommended by the Ministry of Public Health in Cameroon [18]. After onsite HIV testing, residual plasmas were transferred into cryotubes labelled with a specific ID code of the participant, and stored at 0°-8°C. Plasma and accompanying sample sheet were then linked by the ID code. Labeled samples were shipped at the National Reference Laboratory (NRL) following universal standards for transport of plasma specimens [19].

A serial algorithm for HIV screening was used in all PMTCT-site laboratories and at the NRL, following the national algorithm for voluntary HIV testing (Fig 1A). Briefly, a sample was assayed by the first test Determine HIV1/2 (Abbott, Minato-ku-Tokyo, Japan); non-reactive samples were reported as negative while in case of reactivity to test-1, a second test was then used for confirmatory analysis (Oraquick). Onsite, indeterminate HIV results were reported as indeterminate, while specimens with indeterminate/discordant HIV results at the NRL were tested with ELISA (ImmunoComb II HIV 1&2 BiSpot), used as tiebreaker. Residual plasma was stored at -70°C at the NRL for quality control or further testing if necessary.

Fig 1

a. Algorithm for HIV rapid testing during a voluntary and free counseling. b. Algorithm for syphilis rapid testing during a voluntary and free counseling.

Syphilis testing

Syphilis testing was performed in all PMTCT-site laboratories using the Treponema Palladium Hemagglutination assay (TPHA) / Veneral Diseases Research Laboratory (VDRL) as per the manufacturer’s instructions and WHO recommendations (Fig 1B) [20].

Data collection

Socio-demographic and clinical characteristics were collected from consenting pregnant ANC1 attendees by a nurse without altering the normal functional routine of the health facility. After completing the questionnaire, participants were sent to the laboratory where plasma was collected for HIV and syphilis screening tests according to the routine procedure at the site. There were three focal persons per sentinel site, consisting of the director of the health facility, an Antenatal Care (ANC) staff, and a laboratory technician. All received four-day training on the protocol, laboratory techniques related to the surveillance, standard operating procedures of data collection and blood sample collection. For all consecutive surveys, data were collected using a surveillance questionnaire for all eligible and consenting pregnant women and socio-demographic information were collected by trained ANC-PMTCT staff. The questionnaires were maintained at the sentinel sites and transported to the research unit of the National AIDS Control Committee (NACC) and the plasma samples were sent to the NRL for analyses.

Data management and statistical analysis

Data was entered in a computer using the CSPRO version 6.2.0 (U.S Census Bureau, ICF International, and Serpro S.A., Washington, DC 20233–8860, USA). The analyses were performed using STATA/SE version 13.0 (STATACORP, Texas, USA). Pregnant women with inconclusive test results were not considered in the analysis. Those who could not benefit from syphilis testing due to stock out were also excluded from the analysis. Continuous variables were reported by median with Interquartile interval range (25th and 75th percentiles) and by mean and standard deviation, while categorical variables were described as frequencies and percentages. The overall prevalence of HIV/syphilis co-infection, Syphilis infection and HIV infection among pregnant women was estimated with an exact binomial confidence intervals (CI) at 95%. Only data from the 2017 (a subset where women were tested for both HIV in the NRL and Syphilis in PMTCT sites) were used to assessed factors associated with HIV/syphilis co-infection. The main outcome (dependent variable) of our analysis was HIV/syphilis co-infection variable which has 4 categories: category 1 = HIV/syphilis co-infection; category 2 = HIV infection only; category 3 = syphilis infection only; category 4 = no infection. A multivariate multinomial logistic regression was used to investigate the factors associated with HIV/syphilis co-infection among pregnant ANC-1 attendees.

Factors that were associated with HIV/syphilis co-infection in the univariate analysis at 0.25 significance level were considered when developing the final multivariate model. To identify potential risk factors, a multivariate analysis using a backward stepwise elimination method was used. All variables that were significant at 5% in multivariate analysis were kept into the final model. The risk factors for syphilis infection were determined using co-variates fitted in the multi-variable logistic regression analysis: age, HIV infection, marital status, education level, occupation, residence, and parity. A likelihood ratio test was used to check for interactions and in determining the best multi-variable model for the data.

Ethical considerations

Ethical clearance for the surveys was obtained from the Cameroon National Ethics Committee (ref N°2017/03/879/CECNERSH/SP). The privacy of consenting pregnant women and data confidentiality were ensured by the use of ID codes. All participants had signed informed consent without any incentive. HIV tests were offered for free and all women tested positive were placed on ART according to the national guidelines. Confidentiality and privacy of the study subjects was ensured by permanently delinking personal identifiers with subject information.

Results

Description of population in 2009, 2012 and 2017 in Cameroon

Over the 7000 pregnant women targeted, the participation rate was higher in 2017 (2017: 98.0%) and was above 90% in the two other surveys (2009: 94.7%, 2012: 93.2%) (Table 1). Overall, the pregnant women enrolled across the threee surveys were relatively young. In fact, 53% of them were aged between 15–24 years in 2009, almost half (49.3% were aged between 15–24 years) in 2012 and 42.7% were aged between 15–24 years in 2017 (Table 1).The median of age sligthly increase overtime going from 24 years (IQR [20-28]) in 2009 to 26 years [21-30] in 2017.

Table 1

Demographics characteristics among pregnant women aged beyond 15 years old, national sentinel surveillance survey of HIV and syphilis in 2009 (n = 6632), 2012 (n = 6521) and 2017 (n = 6859).

Variable	2009 (n = 6632)		2012 (6521)		2017 (n = 6859)
	Number	Percentage	Number	Percentage	Number	Percentage
Age (Median [IQR])	24 [20–28]		25 [21–29]		26 [21–30]
Age
15–19	1525	23.0	1193	18.3	1033	15.1
20–24	1990	30.0	2024	31.0	1896	27.6
25–29	1592	24.0	1676	25.7	1886	27.5
30–34	928	14.0	1041	16.0	1282	18.7
35–49	597	9.0	587	9.0	762	11.1
Level of education
None	1260	19.0	1071	16.4	949	13.8
Primary	2122	32.0	2319	35.6	1712	24.9
Secondary	2852	43.0	2592	39.8	3275	47.8
University	398	6.0	539	8.3	923	13.5
Occupation
Housewife	3774	56.9	3241	49.7	3385	49.4
Student	889	13.4	926	14.2	1168	17
Employees	1486	22.4	2152	33.0	1604	23.4
Other	484	7.3	202	3.1	702	10.2
Marital status
Single	1240	18.7	NA	NA	1504	21.9
Married / Cohabiting	5326	80.3	NA	NA	5305	77.4
Widow/ Divorced	67	1.0	NA	NA	50	0.7
Area of residence
Urban	3979	60	2787	57.3	3932	57.3
Rural	2653	40	3734	42.7	2927	42.7

Morever half or more of the women enrolled were housewive (57% in 2009; 49.7% in 2012 and 49.4% in 2017) and the majority of them were married or cohabiting (80.3% in 2009 and 77.4% in 2017). Unfortunatly this variable was missed in the 2012 survey. In addition, the proportion of pregnant women who reached university sligthly increased over the year (6% in 2009; 8.3% in 2012 and 13.5% in 2017) and more than half of them were living in urban area in all the three surveys (60% in 2009; 57.3% in 2012 and 57.3% in 2017) (Table 1).

Evolution of Syphilis HIV and co-infection among pregnant women from 2009 to 2017

The overall prevalence of syphilis increased hugely going from 0.6% (95% CI:0.40–0.80) in 2009 to 5.6% (95% CI:4.93–6.40) in 2017 (Fig 2). This increasing trend was also observed for the coinfection HIV/syphilis ranging from 0.05% (95% CI:0.01–0.13) in 2009 to 0.49% (95% CI:0.29–0.76) in 2017. This prevalence is five times higher in 2012 than in 2009 (2.93% vs. 0.6%, p <0.001) and almost nine times higher in 2017 than in 2009 (5.6% vs 0.6%, p<0.001). However, we found a significant decrease in the prevalence of HIV infection from 7.6% (95% CI: 6.99–8.28) in 2009 to 5.7% (95% CI: 4.93–6.4) in 2017.

Fig 2

Evolution of seroprevalence of HIV/syphilis co-infection, HIV and syphilis from 2009 to 2017 in Cameroon.

Description of population and distribution of syphilis and HIV/syphilis co-infection in 2017 in Cameroon

In 2017, a total of 3901 pregnant women (56.9% of the total pregnant women enrolled) were tested for syphilis. Almost half of them (47.9%) were living in urban area and were aged less than 25 years (44.7%). About three fifth (62.9%) attended at least the secondary education level. About three fourth (75.1%) were married or cohabiting, and over two fifths (43.4%) were housewife and 23.9% were nulliparous (Table 2).

Table 2

Demographics characteristics among pregnant women aged beyond 15 years old who were tested for HIV and syphilis, national sentinel surveillance survey of HIV and syphilis 2017.

Characteristic	Overall Population (N = 3901)		Among HIV+ Population (N = 222)
	Number	Percentage	Number	Percentage
HIV Status
HIV positive	222	5.8	NA	NA
HIV negative	3,574	94.2	NA	NA
Total	3796	100.0	NA	NA
Residence
Urban	2,031	52.1	118	53.2
Rural	1,870	47.9	104	46.8
Total	3901	100.0	222	100.0
Region
Adamawa	477	12.2	15	6.8
Centre	559	14.3	49	22.1
East	560	14.4	46	20.7
Far North	365	9.4	8	3.6
Littoral	76	2.0	4	1.8
North	116	3.0	0	0.0
North West	211	5.4	11	5.0
West	240	6.2	6	2.7
South	614	15.7	28	12.6
South West	683	17.5	55	24.8
Total	3901	100.0	222	100.0
Type of union
Single	939	24.6	64	29.5
Married/Cohabiting	2,862	75.1	151	69.6
Widow/Divorced	12	0.3	2	0.9
Total	3813	100.0	217	100.0
Education
None	452	11.7	18	8.2
Primary	981	25.4	71	32.4
Secondary	1,929	49.9	25	11.4
Higher	504	13.0	105	48.0
Total	3866	100.0	219	100.0
Primiparous
Yes	931	23.9	24	10.8
No	2,956	76.1	198	89.2
Total	3887	100.0	222	100.0
Number of pregnancies
0	931	23.9	24	10.9
2 to 3	1,496	38.5	78	35.1
4 to 5	888	22.9	78	35.1
>5	572	14.7	42	18.9
Total	3887	100.0	222	100.0
Age (in years)
<25	1,734	44.7	53	24.0
15–19	665	17.1	7	3.2
20–24	1,069	27.6	46	20.8
≥ 25	2,144	55.3	168	76.0
25–29	1,011	26.1	63	28.5
30–34	721	18.6	59	26.7
35–49	412	10.6	46	20.8
Total	3878	100.0	221	100.0
Occupation
Housewife	1,655	43.4	91	42.3
Student	727	19.0	19	8.8
Formal employee (Civil servant)	308	8.1	17	7.9
Informal employee/entrepreneurship	889	23.3	72	33.5
Unemployed	163	4.3	4	1.9
Others	74	1.9	12	5.6
Total	3816	100.0	215	100.0

NA: Not Applicable

Among the pregnant women who were tested for syphilis, a total of 222 HIV tests results were positive for HIV giving a sero-prevalence of 5.8% of HIV infection, and 24% of them were aged less than 25 years old (Table 2).

According to HIV status, prevalence of syphilis was higher among HIV positive pregnant women than HIV negative (8.6% vs 5.3%, p = 0.049), with a borderline significance (Table 3). Moreover, the prevalence of syphilis was higher in rural area than in urban area (7.2% vs 4.2%, p < 0.0001) and in Southern Regions (Centre, East, Littoral, North West, West and South) than Northern (Adamawa, North and Far North) one (6.2 vs 4.0, p = 0.005), there was no significant difference among HIV + population (8.5% vs 8.7%, p = 1; 8.5 vs 8.7, p = 0.611) according to residence and regions respectively.

Table 3

Prevalence of Syphilis among pregnant women aged beyond 15 years old who were tested for HIV and syphilis by selected demographic characteristics, national sentinel surveillance survey of HIV and syphilis 2017.

Characteristic	Overall Population (N = 3901)							Among HIV+ Population (N = 222)
	Syphilis +		Syphilis -		Total		p-value#	Syphilis +		Syphilis -		Total		p-value#
	n	%	n	%	n	%		n	%	n	%	n	%
HIV Statusb							0.049							NA
HIV positive	19	8.6	203	91.4	222	100.0		NA	NA	NA	NA	NA	NA
HIV negative	191	5.3	3,383	94.7	3574	100.0		NA	NA	NA	NA	NA	NA
Residence							<0.0001							1
Urban	85	4.2	1,946	95.8	2031	100.0		10	8.5	108	91.5	118	100.0
Rural	135	7.2	1,735	92.8	1870	100.0		9	8.7	95	91.4	104	100.0
Region Group							0.005							0.611
Northern	38	4.0	920	96.0	958	100.0		2	8.7	21	91.3	23	100.0
Southern	182	6.2	2,761	93.8	2943	100.0		17	8.5	182	91.5	199	100.0
Type of union							0.372							0.178
Single	59	6.3	880	93.7	939	100.0		8	12.5	56	87.5	64	100.0
Married/Cohabiting/ Widow/Divorced	158	5.5	2,716	94.5	2874	100.0		10	6.5	143	93.5	153	100.0
Education							0.019							0.112
None	22	4.9	430	95.1	452	100.0		3	16.7	15	83.3	18	100.0
Primary	74	7.5	907	92.5	981	100.0		4	5.6	67	94.4	71	100.0
Secondary	101	5.2	1,828	94.8	1929	100.0		6	5.7	99	94.3	105	100.0
Higher	20	4.0	484	96.0	504	100.0		4	16.0	21	84.0	25	100.0
Primiparous							0.172							0.039
Yes	59	6.3	872	93.7	931	100.0		5	20.8	19	79.2	24	100.0
No	161	5.5	2795	94.6	2956	100.0		14	7.1	184	92.9	198	100.0
Number of pregnancies							0.128							0.005
0	59	6.3	872.0	93.7	931	100.0		5	26.3	19	9.4	24	100.0
2 to 3	71	4.8	1,425	95.3	1555	100.0		1	5.3	77	37.9	78	100.0
4 to 5	49	5.5	839	94.5	888	100.0		8	42.1	70	34.5	78	100.0
>5	41	7.2	531	92.8	572	100.0		5	26.3	37	18.2	42	100.0
Age (in years)							0.199							0.258
15–24	104	6.0	1,630	94.0	1734	100.0		2	3.8	51.0	96.2	53	100.0
25–49	114	5.3	2,030	94.7	2144	100.0		17	10.1	151	89.9	168	100.0
Occupation							0.297							0.982
Housewife	83	5.0	1572	95.0	1655	100.0		8	8.8	83	91.2	91	100.0
Student	44	6.1	683	94.0	727	100.0		2	10.5	17	89.5	19	100.0
Formal employee (Civil servant)	13	4.2	295	95.8	308	100.0		1	5.9	16	94.1	17	100.0
Informal employee/entrepreneurship	59	6.6	830	93.4	889	100.0		6	8.3	66	91.7	72	100.0
Unemployed & Others	16	6.8	221	93.3	237	100.0		0	6.3	15	93.8	15	100.0

#: Fisher’s Exact Test

bNot Applicable (NA) in the HIV-syphilis co-infection cross table.

Among pregnant HIV positive women, we found that prevalence of syphilis was higher among nulliparous women than non-nulliparous women (20.8% vs 7.1%, p = 0.039).

Factor associated with Syphilis and HIV/syphilis co-infection in 2017 in Cameroon

The multivariate multinomial logistic regression revealed that residence localization was associated with syphilis infection. After adjusting for the effects of other variables, pregnant women residing in rural areas were more likely to be infected with syphilis than those living in the urban area (aOR = 1.8 [95% CI: 1.3–2.4]) (Table 4). We also found that age and type of union were associated with HIV/syphilis co-infection. In fact, single or unmarried pregnant women were three times more likely to be co-infected by HIV and syphilis than married, cohabiting, widow or divorced pregnant women (aOR = 2.9 [95% CI: 1.0–8.2]). Pregnant women aged 25–49 years were strongly more likely to be co-infected (aOR = 15.1 [95% CI: 3.0–75.7]) by HIV and syphilis than those aged 15–24 years. Furthermore, region; type of union, nulliparous and age were associated to HIV infection. Living in Northern Region (Adamawa, North and Far North) was associated with a lower risk of being infected with HIV (aOR = 0.6 [95% CI: 0.5–0.9]) and syphilis infection (aOR = 0.6 [95% CI: 0.4–0.9]) than living in Southern Regions (Centre, East, Littoral, North West, West and South), but being single or unmarried was associated with a higher risk of HIV infection (aOR = 1.5 [95% CI: 1.1–2.1]) than being in couple (married or not), divorced or widow. In the same way, having 25–49 years old or being multiparous were associated with a higher risk of HIV infection (aOR = 2.7 [95% CI: 1.7–4.3]; aOR = 2.0 [95% CI: 1.4–2.6] respectively).

Table 4

Risk factors associated to HIV/syphilis co-infection among pregnant women aged beyond 15 years old who were tested for HIV and syphilis, national sentinel surveillance survey of HIV and syphilis 2017.

Characteristics	Univariate Analysis			Multivariate Analysis
	HIV/syphilis Co-infection vs No infection	HIV infection vs No infection	Syphilis infection vs No infection	HIV/syphilis Co-infection vs No infection	HIV infection vs No infection	Syphilis infection vs No infection
	OR [95% IC]	OR [95% IC]	OR [95% IC]	aOR [95% IC]	aOR [95% IC]	aOR [95% IC]
Residence
Urban	Ref	Ref	Ref*	Ref	Ref	Ref
Rural	1 [0.4–2.5 ]	0.8 [0.7–1.1 ]	1.9 [1.4–2.5 ]	1.1 [0.4–2.8 ]	0.9 [0.7–1.1 ]	1.8 [1.3–2.4 ] μ
Region Group
Northern	0.3 [0.1–1.4 ]	0.6 [0.4–0.8 ]	0.6 [0.4–0.9 ]	0.5 [0.1–2.1 ]	0.6 [0.5–0.9 ] μ	0.6 [0.4–0.9 ] μ
Southern	Ref	Ref	Ref*	Ref	Ref	Ref
Type of union
Single	2.5 [1–6.4 ]	1.1 [0.8–1.4 ]	1.1 [0.8–1.5 ]	2.9 [1–8.2 ] a	1.5 [1.1–2.1 ] μ	1 [0.7–1.5 ]
Married/Cohabiting/Widow/Divorced	Ref	Ref	Ref*	Ref	Ref	Ref
Education
None	Ref	Ref	Ref*
Primary	0.7 [0.1–3 ]	2.1 [1.3–3.3 ]	1.8 [1.1–3.1 ]
Secondary	0.5 [0.1–1.9 ]	1.4 [0.9–2.3 ]	1.2 [0.7–2 ]
Higher	1.2 [0.3–5.4 ]	1.1 [0.6–2 ]	0.8 [0.4–1.5 ]
Primiparous
Yes	Ref	Ref	Ref*	Ref	Ref	Ref
No	0.9 [0.3–2.6 ]	3 [2.1–4.5 ]	0.9 [0.6–1.2 ]	0.4 [0.1–1.2 ]	2.7 [1.7–4.3 ] μ	1 [0.7–1.4 ]
Number of pregnancies
0	Ref	Ref	Ref*
2 to 3	0.1 [0–1.1 ]	2.6 [1.7–4 ]	0.8 [0.6–1.2 ]
4 to 5	1.8 [0.6–5.5 ]	3.8 [2.5–5.8 ]	0.9 [0.6–1.3 ]
>5	1.7 [0.5–6 ]	3.1 [1.9–4.9 ]	1.2 [0.7–1.8 ]
Age (in years)
15–24	Ref	Ref	Ref*	Ref	Ref	Ref*
25–49	7.2 [1.7–31.2 ]	2.4 [1.9–3.2 ]	0.8 [0.6–1.1 ]	15.1 [3–75.7 ]μ	2 [1.4–2.6 ] μ	0.8 [0.6–1.2 ]
Occupation
Housewife	Ref	Ref	Ref*
Student	0.6 [0.1–2.6 ]	0.4 [0.3–0.6 ]	1.3 [0.8–1.8 ]
Formal employee (Civil servant)	0.7 [0.1–5.4 ]	1.2 [0.7–1.8 ]	0.9 [0.5–1.6 ]
Informal employee/entrepreneurship	1.5 [0.5–4.3 ]	1.6 [1.2–2.2 ]	1.4 [1 – 2 ]
Unemployed & Others	0.9 [0.1–7.4 ]	1.3 [0.8–2.1 ]	1.5 [0.8–2.6 ]

a: attempted a borderline.

Ref: reference category

#: Global p-value < 0.25

*: Global p-value < 0.05

μ: P-value < 0.05

Discussion

The aim of our study was to describe the prevalence trends of syphilis and HIV/syphilis co-infection among pregnant women using sentinel surveillance surveys over a nine-year period and investigate the risk factors associated to HIV co-infection.

To date, maternal syphilis seroprevalence maybe increasing in Cameroon. From 2009 to 2017, the prevalence of syphilis among pregnant women attending ANC in Cameroon increased significantly from 0.6% (95% CI:0.40–0.80) in 2009 to 5.6% (95% CI:4.93–6.40). HIV and syphilis co-infection is also increasing, due to the re-emergence of syphilis among ANC-1 attendees in Cameroon. The prevalence of HIV/syphilis co-infection significatively increased also from 0.05% (95% CI:0.01–0.13) in 2009 to 0.49% (95% CI:0.29–0.76) in 2017. Although the prevalence of HIV/syphilis co-infection is still low (<1%), it was ten times higher in 2017 than in 2009 (0.49% vs 0.05%, p<0.001). In Sub-Saharan African countries, the prevalence of syphilis among pregnant women was estimated to range from 0.6% in Senegal to 14.0% in Equatorial Guinea [21-24]. In 2017, the prevalence of syphilis found among pregnant women attending antenatal clinics in Cameroon was among the middle’s ones in Sub-Saharan African countries (5.6%). The prevalence of syphilis among pregnant women in Cameroon was higher in rural than in urban areas. The same result was found in Tanzania where syphilis prevalence was highest among pregnant women from remote rural residents (16.0%) compared to urban residents (7.0%) [25]. In contrast, a study conducted in Zambia found the opposite and established that syphilis prevalence was highest among pregnant women residing in urban residence (9.2%) as compared to those residing in rural (7.8%) [27].

Moreover, the prevalence of syphilis was higher among HIV positive pregnant women than HIV negative (8.6% vs 5.3%). Similar result has also been found in Rwanda in 2011, where syphilis prevalence was highest among HIV-positive pregnant women (10.8%) compared to HIV negative pregnant women (1.8%) [9]. This could due to the fact that pregnant women living with HIV may have a weaker immune system than other pregnant women. Preventives strategies should prioritize this group of the population.

In contrast of syphilis trend, our study found a significantly decrease of HIV infection among pregnant women from a prevalence of 7.6% (95% CI: 6.99–8.28) in 2009 to 5.7% (95% CI: 4.93–6.4) in 2017. We observe the same trend by triangulate with DHIS data collected routinely among pregnant women in the country. This trend may be the result of several strategies implemented by the country to strengthen the prevention of HIV particularly among pregnant women [28-30].

Living in a rural area was a risk factor for syphilis infection. This can be explaining by the fact that some pregnant women do not attend ANC services early and some of the rural area facilities may not have syphilis test permanently for screening pregnant women. Furthermore, in this area people may not use condom frequently and may not aware of its importance. As recommended in the WHO guide, syphilis (new or old infection) is cure in first intention with benzathine penicillin [20]. Thus the global stockout of benzathine penicillin in 2016 could have play a role in the sero-prevalence rate and the spread of syphilis, particularly in rural areas [30]. We also found that living in Northern regions (Adamawa, North and Far North) was associated with a lower risk for both syphilis infection as well as HIV infection. Southern regions have the most affected regions which include South, East, Centre and North-West. It is worthy to note that Northern regions are more muslim and southern ones are more Christian. These regions should therefore be considered for priority interventions to curb this high HIV and syphilis burden.

As found in other studies [9, 27], being single and being aged beyond 25 years old were associated with a higher risk of HIV/syphilis co-infection and HIV infection. Possible reasons that may explain these results include that single women may have several sexual partners before moving in a stable relationship and due to their economic vulnerability, they are more involved in transactional sex which increases their exposure to HIV and syphilis as a result [31-33]. Women aged beyond 25 years old and multiparous were more likely to be exposed to HIV and syphilis longer than younger, since they were sexually active.

The strength of our study is the representative sample size which covered the whole Regions of the country and offers the possibility to generalize our results [27].

This study has some limitations. Several sexual risk factors were not collected in these surveys and therefore more data are needed on sexual behaviors and sexual partners experience. Data were pooled across all sites for analysis, and therefore site-level variation was not accounted for in our analysis. This may have impacted our trends assessment but we believe that these limitations did not significantly affect the final interpretation of the overall trend of our study findings. The funding allocated to the project was not enough to provide syphilis tests at the study sites, neither at the NRL. Then syphilis test was done in sentinel sites which had the test for their routine ANC services during study period. Therefore, all pregnant women enrolled in our surveys were not tested for syphilis due to stock out at the level of some sentinel sites. This might have slightly affected the power of our analyses by reducing the sample size of analyses, and some participants who might be positive for syphilis infection were not included in this analysis. In addition, information on ART for the women who were tested positive to HIV were not collected. This could be used to improve future Sentinel Surveillance Surveys and the WHO protocol on this aspect in the area of Test and Treat. Moreover, the titres were measured for VDRL for the majority of the sentinel sites to determine if it was a current infection or an old or scarring infection, but this information was not collected during the survey. This could have helped to further refine our factor analysis of the re-emergence of syphilis cases in the country.

Conclusion

Antenatal care provides an excellent opportunity to screen women for infections which are common and treatable and can be transmitted vertically. HIV and syphilis infections remain common in pregnant women. Sentinel surveillance surveys provide a good opportunity to collect data which could provide useful information for decision making, especially in the era of dual elimination (HIV and syphilis). The data of these surveys show that syphilis is becoming a major public health problem in Cameroon. The trends of syphilis among pregnant women confirms a growing burden of syphilis in the general population of Cameroon, and the need to reinforce surveillance and prevention strategies to fight STIs. To control syphilis infection, we need to involve keys actors like clinicians, because they must educate patients, counsel them in sexual risk reduction, and routinely screen those at increased risk. Finally, there is a need of a huge emphasizing of systematic screening of STIs particularly among people living with HIV. Strategies such as political advocacy of systematic counseling and testing of syphilis at ANC and treatment could be valuable. These strategies may target adult pregnant women, who are HIV-positive, single living in rural area priority in Southern Cameroon (South, East, Centre and North-West regions).

(DTA)

Click here for additional data file.

8 Jul 2020

PONE-D-20-11194

Highlighting a population-based re-emergence of Syphilis infection and assessing associated risk factors among pregnant women in Cameroon: Evidence from the 2009, 2012 and 2017 national sentinel surveillance surveys of HIV and syphilis.

PLOS ONE

Dear Dr. KENGNE-NDE,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Aug 22 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Remco PH Peters, MD, PhD, DLSHTM

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0208963

- https://journals.sagepub.com/doi/10.1177/0956462415624058

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

3.  We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

4. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

**********

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

Reviewer #1: No

Reviewer #2: No

Reviewer #3: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

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: Thank you for the opportunity to review this manuscript. The manuscript a very important issue in public health and in the efforts towards the dual elimination of mother to child transmission of syphilis. The paper is generally well written but I think that the methods have not been sufficiently described or are inadequate to warrant the conclusions made. The authors conclude that there was a massive increase in syphilis prevalence among first ANC clinic attendees without providing sufficient evidence to exclude sampling issues, testing procedures, and errors in the analysis e.g. lack of weighting in the analysis. I have detailed my comments to the authors below

Abstract

• Line 3: This sentence should be syphilis and HIV can be transmitted from and not by

• Lines 8- 14: you need to clarify that the survey was conducted at the same sites every time and how many were enrolled in each survey.

• Lines 27-28: Please use word as suggest and not confirm as you haven’t provided evidence that syphilis has been re-emerging in Cameroonian general population besides the findings from the surveys

Introduction

• Line 33: sentence should read “Sub-Saharan African”. Please change this here and elsewhere where you have used this term – see line 54

• Line 42: remove “HIV and” and write sexual transmitted as sexually transmitted

• Line 67: in the methods, you refer to a nine year period and here to an eight. Please clarify

Methods

• Generally the methods need to be improved.

• Lines 70- 88: The design should be a secondary analysis of data from three ANC sentinel surveys. There after you need to put an extra section on the description of the three surveys. A table mighty be useful to highlight differences and similarities in the survey methods used across the 3 surveys Some important information to include is

o How often are the surveys done? Are they done on a regular basis or as needed?

o You say the surveys included 20 sentinel sites in 10 regions. Were these sentinel sites the same across all three surveys? How are the sentinel sites selected?

o You also say you included 10 surveillance health facilities. What is the nature of the surveillance health facilities and how do they differ or how are they related to the sentinel sites?

o What is the nature of a sentinel site – is it a clinic or hospital

• Lines 89- 110: This should be a sub-section of the proposed section on description of the surveys. Was syphilis testing conducted in all three surveys? If so how was it done in each if the survey? Later in the methods section (line 131- 134) you mentioned that only the 2017 did you have women tested for both HIV and syphilis at PMTCT sites. It’s not clear what this means? Where were the labs located in relation to the sentinel sites?

• Lines 124- 142: You did not define what your outcomes of the analysis are. Does the outcome have more than 2 responses? If yes which ones? This would justify use of the multinomial logistic regression.

• Since the data were collected in a survey where there might be clustering of outcomes, was the data weighted? Were the same data weights used across the 3 surveys? If no weights were applied, I would recommend a weighted analysis for calculating the survey specific syphilis sero-prevalence’s and even the analyses of trends across the surveys or at least some justification for why a weighted analysis is not necessary

Results

• Lines 151- 175: Please combine tables 1a, b and c to make it easier to compare the characteristics of participants across the surveys. Why not categorise age into fewer categories – 15- 24, 25- 35, 35- 49. Why were variable available different across the surveys?

• Lines 176- 183: Did you do a trend analysis? How? Please describe in the methods

• Lines 184: Figure 2 is not clear at all. A simple bar graph with error bars could be clearer

• Line 186- 192: what % of those enrolled in the 2017 survey did the 3901 tested for syphilis represent? Why were only these ones tested for syphilis? Please the information in the methods

• Line 196: please add the numbers for the total population and HIV positive population to table 2

• Also are the results in Table 2 weighted?

• Did you collect information on ART for the women who were HIV POSITIVE?

Discussion

• Line 248 – add HIV to co-infection

• Line 249- 253: I would tone down this assertion that syphilis prevalence is increasing given the methodological issues highlighted. Perhaps say maternal syphilis seroprevalence maybe increasing

• Have you looked at other data sources on maternal seropositivity e.g. from DHIS that you can triangulate with

• Line 259: not sure what middles in this sentence means

• Line 266- 269: you don’t discuss reasons why syphilis is higher in HIV positive women and how future surveys or studies can untangle this. The HIV/syphilis positive women – are they more likely to be on ART? Are they not on ART?

• Line 274: what was ANC attendance like in rural areas and has it changed over the 8/9 year period here? You don’t present rural urban distribution in the 2009 and 2012 surveys? It is possible that the increased prevalence is due to more rural women taking part in the survey?

Reviewer #2: This is an interesting study that provides a lot of food for thought: how can syphilis explode while HIV declines? Fascinating.

I have a few comments/suggestions, mostly minor but one major.

Minor

1. Did availability of antibiotics (e.g. over the counter purchases) change between the surveys?

2. line 57-59. This is stating the same thing twice. Also the causal link between syphilis and HIV is not realy proven, so please weaken your statement (e.g. syphilis has been implicated in susceptibility to HIV)

3. line 272. Please change "is the result" to "may be the result"

4. line 279. Is region associated with religion? If so please mention this.

5. While the ms is generally well written a few sentences seem to be not standard English. While acceptable it might be useful to have a native speaker check its grammar.

6. Were the same ANC sites used in the three surveys? This also impacts analysis

Major

1. Statistical analysis seems to ignore the structure/design of the survey which looks more like a multi-stage survey than a simple random sample. STATA offers excellent routines for analysing this type of data. Same applies to sample size calculation.

2. Perhaps the different logistic regression analyses can be applied to the all three surveys?

3. The increase in syphilis should also be demonstrated using logistic regression with survey year as one of the covariables. One can thereby adjust for changes in other risk factors.

Reviewer #3: The purpose of this article is to monitor changes in the seroprevalence of HIV/syphilis co-infection and syphilis infection and associated risk factors in Cameroon from 2009 to 2012 and 2017. These questions are important as they provide evidence for interventions for the prevention and control of HIV/AIDS and syphilis. To carry-out the objectives, the authors use cross-sectional antenatal care surveys conducted in 2009, 2012, and 2017 from 20 sentinel surveillance sites across 10 regions of Cameroon. The authors found the following:

1) HIV/syphilis co-infection increased from 0.05% in 2009 to 0.49% in 2017. Pregnant women aged 25–49 compared with those aged 15–24 were 15.1 times more likely to be co-infected. Single or unmarried compared to those who were married, cohabitating, widowed, or divorced were 2.9 times more likely to have a co-infection.

2) Syphilis infection increased from 0.6% in 2009 to 5.6% in 2017. Pregnant women living in rural areas were 1.8 times more likely to contract syphilis than those in urban areas. Pregnant women living in Northern Region were 0.6 times as likely to have a syphilis infection than those in the Southern Regions.

3) HIV infection decreased from 7.6% in 2009 to 5.7% in 2017. Pregnant women aged 25–49 compared with those aged 15–24 were 2.7 times more likely to be HIV positive. Multiparous pregnant women 2.0 times more likely to have an HIV infection than non-nulliparous.

This work provides evidence for national policy, resource allocation, and the evidence base for interventions for the control and prevention of HIV and syphilis in Cameroon and the global elimination of mother-to-child transmission of HIV and syphilis.

Comments/Questions:

1. In the methods section, please specify the sampling method used. Specify probability or nonprobability sampling and type of sampling (ie. simple random, systematic, convenience, …) that was used.

2. Did the analysis have 2 or more dependent or outcome variables or were there multiple independent or response variables? The methods mention multivariate and multivariable interchangeably.

3. The results sections is bit hard to follow. Consider grouping results by the specific infection and co-infection so that the reader can look to the results to inform the story that is being told in the discussion section.

4. On line 156, what is meant by the term ‘monogomous regime’?

5. “Enroled’ (British English) and ‘enrolled’ (American English) are used interchangeably throughout the paper. Choose one or the other according to the journal’s style guide.

6. Also check for capitalization where it is not needed (ie. see line 270 “In contrast of Syphilis trend”, line 279, …).

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Tendesayi Kufa

Reviewer #2: Yes: Nico Nagelkerke

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

18 Sep 2020

Editor

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

Authors: Our manuscript meets PLOS ONE's style requirements, including those for file naming, Thank you.

2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0208963

- https://journals.sagepub.com/doi/10.1177/0956462415624058

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

Authors: In our revision we cite or rephrase duplicated text outside the methods section. Thank you.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

Authors: According to the national survey data sharing policies dictated by the ethic committee, access to data of 2009 and 2012 data survey is not systematic and is subject to a request via infos@cnls.cm; Phone: (00237) 222 22 57 58, Fax: 222 23 34 39; P.O BOX: 1459 Yaounde, Cameroon. Nevertheless, data for 2017 survey is not submitted to this restriction and we submitted it with this revised manuscript. Thank you.

4. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:.” as necessary).

Authors: The list of authors has been amended. Each author is linked to an affiliation. Thank you.

Reviewer #1:

Thank you for the opportunity to review this manuscript. The manuscript a very important issue in public health and in the efforts towards the dual elimination of mother to child transmission of syphilis. The paper is generally well written but I think that the methods have not been sufficiently described or are inadequate to warrant the conclusions made. The authors conclude that there was a massive increase in syphilis prevalence among first ANC clinic attendees without providing sufficient evidence to exclude sampling issues, testing procedures, and errors in the analysis e.g. lack of weighting in the analysis. I have detailed my comments to the authors below.

Authors: We thank the Reviewer 1 for this summary.

Abstract

• Line 3: This sentence should be syphilis and HIV can be transmitted from and not by

Authors: Thank you for this comment. We modified the sentence in the manuscript.

• Lines 8- 14: you need to clarify that the survey was conducted at the same sites every time and how many were enrolled in each survey.

Authors: Thank you for this comment. We clarified in the manuscript.

• Lines 27-28: Please use word as suggest and not confirm as you haven’t provided evidence that syphilis has been re-emerging in Cameroonian general population besides the findings from the surveys.

Authors: Thank you for this comment. We used the word “suggest” in the manuscript.

Introduction

• Line 33: sentence should read “Sub-Saharan African”. Please change this here and elsewhere where you have used this term – see line 54

Authors: Thank you for this comment. We changed the term “Sub-Saharan” with “Sub-Saharan African” here and elsewhere we have used it in the manuscript.

• Line 42: remove “HIV and” and write sexual transmitted as sexually transmitted

Authors: Thank you for this comment. We modified the sentence in the manuscript.

• Line 67: in the methods, you refer to a nine-year period and here to an eight. Please clarify

Authors: Thank you for this remark. We corrected the error to a “nine-year period” in the manuscript.

Methods

• Generally, the methods need to be improved.

• Lines 70- 88: The design should be a secondary analysis of data from three ANC sentinel surveys. There after you need to put an extra section on the description of the three surveys. A table mighty be useful to highlight differences and similarities in the survey methods used across the 3 surveys Some important information to include is:

Authors: We thank reviewer 1 for this suggestion. Since the surveys were very similar across the year, we specified details in the paragraph of the “Description of the surveys, study design and participants” section. Thank you.

o How often are the surveys done? Are they done on a regular basis or as needed?

Authors: The survey are supposed to be done in a Regular basis (2 years). But, according to funds availability, one survey for a specific year could be postponed. Thank you.

o You say the surveys included 20 sentinel sites in 10 regions. Were these sentinel sites the same across all three surveys? How are the sentinel sites selected?

Authors: Thank you for this important question which can help others authors in the reproductibility of this study. The sentinel sites were the same across all the three surveys. As already described in the manuscript (Line 76 – 79), The sentinel sites were chosen based on their capacity to provide both ANC and PMTCT services, their location (urban and rural settings in each region of the country), ANC attendance (capacity to enrol at least 300 pregnant ANC1 attendees during the study period of three months).

o You also say you included 10 surveillance health facilities. What is the nature of the surveillance health facilities and how do they differ or how are they related to the sentinel sites?

Authors: Thank you for this question. As described in the manuscript (line 73-76), we conducted the surveys in 20 sentinel sites that included 60 HIV surveillance health facilities (routine collection points). So, each sentinel site has 3 HIV surveillance health facilities. The surveillance health facilities were health facilities from public and private sector. The collection point was found in a sentinel site (related to a specific geographic area of the region). These collection points are hospitals with high ANC attendance that meet the selection criteria as recommended by the WHO.

o What is the nature of a sentinel site – is it a clinic or hospital

Authors: A sentinel site is a specific geographic area in the region. In the sentinel site, HIV surveillance health facilities (clinic and or hospital) were the data collection points. Each sentinel site had 3 data collection points. Thank you.

• Lines 89- 110: This should be a sub-section of the proposed section on description of the surveys. Was syphilis testing conducted in all three surveys? If so how was it done in each if the survey?

Authors: Yes, the syphilis testing was conducting in all three surveys. It was done using the Treponema Palladium Hemagglutination assay (TPHA) / Veneral Diseases Research Laboratory (VDRL) as per the manufacturer’s instructions and WHO recommendations. Thank you.

Later in the methods section (line 131- 134) you mentioned that only the 2017 did you have women tested for both HIV and syphilis at PMTCT sites. It’s not clear what this means?

Authors: HIV testing was conducted for women at the sites and at the reference laboratory. Results from the reference laboratory were used. Be it 2009, in 2012, Syphilis test was performed consistently at the level of the PMTCT sites using the Treponema Palladium Hemagglutination assay (TPHA) / Veneral Diseases Research Laboratory (VDRL) and results were synthesized at the national level. Thank you.

Where were the labs located in relation to the sentinel sites?

Authors: The labs were found in each of the PMTCT sites and this was a condition to select that site as a surveillance health facility. The reference lab was found in the capital of the country (Yaounde) and tested all the samples collected from sentinel sites. Thank you.

• Lines 124- 142: You did not define what your outcomes of the analysis are. Does the outcome have more than 2 responses? If yes which ones? This would justify use of the multinomial logistic regression.

Authors: The main outcome of our analysis was HIV/syphilis co-infection variable which has 4 responses:

Response 1 = HIV/syphilis co-infection

Response 2 = HIV infection only

Response 3 = Syphilis infection only

Response 4 = No infection

We specify this in the method section (Line 141).

Thank you.

• Since the data were collected in a survey where there might be clustering of outcomes, was the data weighted? Were the same data weights used across the 3 surveys? If no weights were applied, I would recommend a weighted analysis for calculating the survey specific syphilis sero-prevalence’s and even the analyses of trends across the surveys or at least some justification for why a weighted analysis is not necessary.

Authors: Based on the WHO protocol for HIV Sentinel Surveillance Survey (Reference 17 in the manuscript) we have implemented a non-probabilistic approach which consisted of a systematic sampling method with two stages

(1) Selection of sentinel sites / surveillance health facilities (routine collection points)

(2) Selection of pregnant women in each study sentinel site. Briefly, study sites were selected by based on: (a) representativeness at regional level, (b) geographical location in each region (urban or rural), (c) availability of PMTCT services and data management system, (d) functionality of the site (staff and materials for ANC/PMTCT, laboratory services and a cold chain), and (e) site willingness for participation into the study. Selected sites were from health facilities of the primary, secondary and tertiary healthcare levels.

However, since the sample is not representative of the population, we think that it is not necessary to have a weighted analysis approach since the objective of WHO sentinel studies is to have a trend of the epidemic and not to extrapolate the results.

Please feel free to find more details in the reference below [1]:

[1] « WHO | Guidelines for conducting HIV sentinel serosurveys among pregnant women and other groups », WHO, mars 21, 2020. https://www.who.int/hiv/pub/surveillance/anc_guidelines/en/ (consulté le mars 21, 2020).

Thank you.

Results

• Lines 151- 175: Please combine tables 1a, b and c to make it easier to compare the characteristics of participants across the surveys.

Authors: The tables 1a, b and c have been combined into one (table 1 line169), Thank you.

Why not categorise age into fewer categories – 15- 24, 25- 35, 35- 49.

Authors: We harmonize the categories of age in the three survey. We considered 15-19 and 20-24 to separate adolescent (15-19) and teenager (20-24), Thank you.

Why were variable available different across the surveys?

Authors: The variable available were different across the surveys because the questionnaire have slightly changed from one survey to another according to the UNAIDS, WHO and the Country strategies and priority. Thank you.

• Lines 176- 183: Did you do a trend analysis? How? Please describe in the methods

Authors: No, we did not do a trend analysis, but we estimated according to the WHO protocol each survey’s seroprevalence and the Confidence Interval that allowed the comparisons to be made. Thank you.

• Lines 184: Figure 2 is not clear at all. A simple bar graph with error bars could be clearer

Authors: We have tried to improve the quality of this graphic and we plead the reviewer 1 to accept the current version. Thank you.

• Line 186- 192: what % of those enrolled in the 2017 survey did the 3901 tested for syphilis represent?

Authors: The 3901 tested for syphilis represented 56.9% of those enrolled in the 2017 survey. This has been had in the manuscript. Thank you.

Why were only these ones tested for syphilis? Please the information in the methods

Authors: Thank you for this important question. Other pregnant women were tested but those with inconclusive results were not considered in the analysis. Those who could not benefit from syphilis testing due to stock out were also excluded from the analysis. The information has been added in the methods section already.

• Line 196: please add the numbers for the total population and HIV positive population to table 2

Authors: We added the numbers for the total population and HIV positive population to table 2. Thank you.

• Also are the results in Table 2 weighted?

Authors: No, the results in Table 2 are not weighted according to the non-probabilistic approach implemented. Thank you.

• Did you collect information on ART for the women who were HIV POSITIVE?

Authors: No, we did not collect this information since it is not included in the WHO protocol.

This could be used to improve the WHO protocol on this aspect in the area of Test and Treat

We use this contribution to improve the discussion part on this aspect. Thank you.

Discussion

• Line 248 – add HIV to co-infection

Authors: We added HIV to co-infection. Thank you.

• Line 249- 253: I would tone down this assertion that syphilis prevalence is increasing given the methodological issues highlighted. Perhaps say maternal syphilis seroprevalence maybe increasing

Authors: Thank you to the reviewer for this. We modified the sentence accordingly.

• Have you looked at other data sources on maternal seropositivity e.g. from DHIS that you can triangulate with

Authors: No, we did not triangulate with DHIS data because the country adopted DHIS recently (2015) and is not collecting Syphilis data routinely as from now. But for HIV we observed the decreases of Seropositivity like in Sentinels surveillance surveys. DHIS is not used now to collect all data (Syphilis, hepatitis, etc.). Nevertheless, it could be really interesting to use these data if collected routinely trough DHIS to monitor and triangulate data of these disease in the future if we include this to DHIS and reduce cost of these survey. We use this contribution to improve the discussion part on this aspect. Thank you.

• Line 259: not sure what middles in this sentence means

Authors: Middles was used to range the seroprevalence found in the country: not small and not high comparing to the ones found in other Sub-Saharan African countries. We modified the sentence to make it clear. Thank you.

• Line 266- 269: you don’t discuss reasons why syphilis is higher in HIV positive women and how future surveys or studies can untangle this. The HIV/syphilis positive women – are they more likely to be on ART? Are they not on ART?

Authors: Thank you to reviewer 1 for this interesting question. Unfortunately, we did not collect information about ART. Nevertheless, pregnant women living with HIH may have a weaker immune system. We use this contribution to improve the discussion section on this point. Thank you.

• Line 274: what was ANC attendance like in rural areas and has it changed over the 8/9 year period here? You don’t present rural urban distribution in the 2009 and 2012 surveys? It is possible that the increased prevalence is due to more rural women taking part in the survey?

Authors: We added rural urban distribution in the 2009 and 2012 surveys in Table 1 (Line 170), and about two fifth of women enrolled were living in urban area in all the three surveys (40% in 2009; 42.7% in 2012 and 42.7% in 2017). We can thus conclude that the increased prevalence is not due to more rural women taking part in the survey. Thank you.

Reviewer #2:

This is an interesting study that provides a lot of food for thought: how can syphilis explode while HIV declines? Fascinating.

Authors: We thank the Reviewer 2 for this appreciation.

I have a few comments/suggestions, mostly minor but one major.

Minor

1. Did availability of antibiotics (e.g. over the counter purchases) change between the surveys?

Authors: No, availability of antibiotics did not change between the surveys. Thank you.

2. line 57-59. This is stating the same thing twice. Also the causal link between syphilis and HIV is not realy proven, so please weaken your statement (e.g. syphilis has been implicated in susceptibility to HIV)

Authors: We thank the reviewer 2 for this remark. We modified our statement accordingly.

3. line 272. Please change "is the result" to "may be the result"

Authors: We thank the reviewer 2 for this comment. We modified our statement accordingly.

4. line 279. Is region associated with religion? If so, please mention this.

Authors: Yes, in the country (Cameroon) Northern regions are most muslim and southern ones are more Christian. We mention this in the discussion section. Thank you.

5. While the ms is generally well written a few sentences seem to be not standard English. While acceptable it might be useful to have a native speaker check its grammar.

Authors: The suggestion have been considered. Thank you.

6. Were the same ANC sites used in the three surveys? This also impacts analysis

Authors: Yes, the sentinel surveillance sites were the same in the three surveys, Thank you.

Major

1. Statistical analysis seems to ignore the structure/design of the survey which looks more like a multi-stage survey than a simple random sample. STATA offers excellent routines for analysing this type of data. Same applies to sample size calculation.

Authors: Based on the WHO protocol for HIV Sentinel Surveillance Survey (Reference 17 in the manuscript) we have implemented a non-probabilistic approach which consisted of a systematic sampling method with two stages

(1) Selection of sentinel sites / surveillance health facilities (routine collection points)

(2) Selection of pregnant women in each study sentinel site. Briefly, study sites were selected by based on: (a) representativeness at regional level, (b) geographical location in each region (urban or rural), (c) availability of PMTCT services and data management system, (d) functionality of the site (staff and materials for ANC/PMTCT, laboratory services and a cold chain), and (e) site willingness for participation into the study. Selected sites were from health facilities of the primary, secondary and tertiary healthcare levels.

However, since the sample is not representative of the population, we think that it is not necessary to have a weighted analysis approach since the objective of WHO sentinel studies is to have a trend of the epidemic and not to extrapolate the results.

Please feel free to find more details in the reference below [1]:

[1] « WHO | Guidelines for conducting HIV sentinel serosurveys among pregnant women and other groups », WHO, mars 21, 2020. https://www.who.int/hiv/pub/surveillance/anc_guidelines/en/ (consulté le mars 21, 2020).

Thank you.

2. Perhaps the different logistic regression analyses can be applied to the all three surveys?

Authors: We limited our analysis to logistic regression in 2017 to have more recent associative factors to guide decision making and strategies more efficiently. Thank you.

3. The increase in syphilis should also be demonstrated using logistic regression with survey year as one of the covariables. One can thereby adjust for changes in other risk factors.

Authors: Thank you for this suggestion. But we think that it could not be right to use the survey year using our logistic regression since we use only the 2017 data survey in the model. It could have been interesting to do so if we did a generalized logistic regression model (with mixed effect or GEE approach), but even in this case it will also challenging to implement since the participants were not the same from one-year study to another. Thank you.

Reviewer #3:

The purpose of this article is to monitor changes in the seroprevalence of HIV/syphilis co-infection and syphilis infection and associated risk factors in Cameroon from 2009 to 2012 and 2017. These questions are important as they provide evidence for interventions for the prevention and control of HIV/AIDS and syphilis. To carry-out the objectives, the authors use cross-sectional antenatal care surveys conducted in 2009, 2012, and 2017 from 20 sentinel surveillance sites across 10 regions of Cameroon. The authors found the following:

1) HIV/syphilis co-infection increased from 0.05% in 2009 to 0.49% in 2017. Pregnant women aged 25–49 compared with those aged 15–24 were 15.1 times more likely to be co-infected. Single or unmarried compared to those who were married, cohabitating, widowed, or divorced were 2.9 times more likely to have a co-infection.

2) Syphilis infection increased from 0.6% in 2009 to 5.6% in 2017. Pregnant women living in rural areas were 1.8 times more likely to contract syphilis than those in urban areas. Pregnant women living in Northern Region were 0.6 times as likely to have a syphilis infection than those in the Southern Regions.

3) HIV infection decreased from 7.6% in 2009 to 5.7% in 2017. Pregnant women aged 25–49 compared with those aged 15–24 were 2.7 times more likely to be HIV positive. Multiparous pregnant women 2.0 times more likely to have an HIV infection than non-nulliparous.

This work provides evidence for national policy, resource allocation, and the evidence base for interventions for the control and prevention of HIV and syphilis in Cameroon and the global elimination of mother-to-child transmission of HIV and syphilis.

Authors: We really appreciate the summary did by the reviewer 3. Thank you.

Comments/Questions:

1. In the methods section, please specify the sampling method used. Specify probability or nonprobability sampling and type of sampling (ie. simple random, systematic, convenience, …) that was used.

Authors: Based on the WHO protocol for HIV Sentinel Surveillance Survey (Reference 17 in the manuscript) we have implemented a non-probabilistic approach which consisted of a systematic sampling method with two stages

(1) Selection of sentinel sites / surveillance health facilities (routine collection points)

(2) Selection of pregnant women in each study sentinel site. Briefly, study sites were selected by based on: (a) representativeness at regional level, (b) geographical location in each region (urban or rural), (c) availability of PMTCT services and data management system, (d) functionality of the site (staff and materials for ANC/PMTCT, laboratory services and a cold chain), and (e) site willingness for participation into the study. Selected sites were from health facilities of the primary, secondary and tertiary healthcare levels.

We specify this in the method section.

Thank you.

2. Did the analysis have 2 or more dependent or outcome variables or were there multiple independent or response variables? The methods mention multivariate and multivariable interchangeably.

Authors: The dependent variable of our analysis was HIV/syphilis co-infection which has 4 responses:

Response 1 = HIV/syphilis co-infection

Response 2 = HIV infection only

Response 3 = Syphilis infection only

Response 4 = No infection

We specify this in the method section.

Thank you.

3. The results sections is bit hard to follow. Consider grouping results by the specific infection and co-infection so that the reader can look to the results to inform the story that is being told in the discussion section.

Authors: We thank the reviewer 3 for this suggestion. We combined Table 1a, b and C into one table to make it easier to the reader. Thank you.

4. On line 156, what is meant by the term ‘monogomous regime’?

Authors: “Monogomous regime” means to be married with one partner. This have been remove to harmonise with the results of other years. Thank you.

5. “Enroled’ (British English) and ‘enrolled’ (American English) are used interchangeably throughout the paper. Choose one or the other according to the journal’s style guide.

Authors: We harmonized and use enrolled (American English). Thank you.

6. Also check for capitalization where it is not needed (ie. see line 270 “In contrast of Syphilis trend”, line 279, …).

Authors: We checked for capitalization where it is not needed, Thank you.

9 Oct 2020

PONE-D-20-11194R1

Highlighting a population-based re-emergence of Syphilis infection and assessing associated risk factors among pregnant women in Cameroon: Evidence from the 2009, 2012 and 2017 national sentinel surveillance surveys of HIV and syphilis.

PLOS ONE

Dear Dr. KENGNE-NDE,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

I would be happy to accept your manuscript for publication once you have addressed the reviewer's minor comments.

==============================

Please submit your revised manuscript by Nov 23 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Remco PH Peters, MD, PhD, DLSHTM

Academic Editor

PLOS ONE

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. 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: Thank you for the opportunity to review this revised manuscript. The authors have addressed most of the comments I had on initial review. The paper reads much better than the initial version and the methods section clearer. However in addressing these, new comments have arisen. I have listed them below:

Abstract

Line 9: add each year

Line 13: add outcomes of the multinomial logistic regression

Introduction

Line 47 – the authors need to add the phrase “such as ” before HIV and syphilis

Line 53- delete the words the highest risk. It sounds repetitive with most in that sentence

Lines 60- 63: the authors don’t address why syphilis is an important co-infection in HIV + patients. Does it have an effect on HIV progression

Methods

Lines 73- 89: The authors clarified what constituted a sentinel site – health facility including its 3 testing points. This is not clear in clear in this paragraph

Lines 112- 115 syphilis testing – how was a case of syphilis decided. Please include the algorithm like you have for HIV. Was the algorithm reverse or traditional. Were titres measured for VDRL to determine current vs old infection

Lines 130- Is the CSPRO software a registered brand? Add developer, city and country

Line 138 – the authors refer to a sub-set to be tested for syphilis. How was this subset selected. Later on they state that shortage of test kits meant sites with no test kits could not be selected for the analysis.

Results

Line 163 – please change from involvement to particioation

Table 1- present medians (IQR) as well as age in cvategories

Table 2 – present numbers before percentages

Table 3: for overall population and those HIV positive add the values of N- the totals

Also primiparous and number of pregnancies overlap as variables. Primiparous and # pregnancies =0 are one and the same thing.

Table 4: see comment above

Fig 1: add syphilis testing algorithm

Discussion

Lines 278- 286: the authors have not addressed the issue of access to treatment as a factor that could explain high rates of syphilis in rural areas. From about 2016, there has been a global benzathine penicillin shortage. Please comment on this

Lines 296- 308: The list of limitations is incomplete. Because selection of sites for syphilis testing were not random, discuss that it was possible that sites which higher syphilis rates could have been included in the survey

Reviewer #2: No further concerns. xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Tendesayi Kufa

Reviewer #2: Yes: Nico Nagelkerke

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

22 Oct 2020

Reviewer #1:

Thank you for the opportunity to review this revised manuscript. The authors have addressed most of the comments I had on initial review. The paper reads much better than the initial version and the methods section clearer. However, in addressing these, new comments have arisen. I have listed them below:

Authors: We thank the Reviewer 1 for this appreciation.

Abstract

• Line 9: add each year

Authors: We added each year in the sentence in the manuscript, Thank you.

• Line 13: add outcomes of the multinomial logistic regression

Authors: We added outcomes of the multinomial logistic regression in the manuscript, Thank you.

Introduction

• Line 47 – the authors need to add the phrase “such as” before HIV and syphilis

Authors: We added “such as” before HIV and syphilis in the manuscript. Thank you.

• Line 53- delete the words the highest risk. It sounds repetitive with most in that sentence

Authors: The words the highest have been deleted. Thank you.

• Lines 60- 63: the authors don’t address why syphilis is an important co-infection in HIV + patients. Does it have an effect on HIV progression

Authors: Thank you to reviewer 1 for this important comment. We have modified the sentence by changing “Co-infection” with “risk factor” since there is no scientific evidence according to our knowledge on the direct influence of syphilis on the progression of HIV.

Methods

• Lines 73- 89: The authors clarified what constituted a sentinel site – health facility including its 3 testing points. This is not clear in clear in this paragraph

• Authors: We clarified more in the manuscript. Thank you.

• Lines 112- 115 syphilis testing – how was a case of syphilis decided. Please include the algorithm like you have for HIV. Was the algorithm reverse or traditional. Were titres measured for VDRL to determine current vs old infection

Authors: Thank you to reviewer for this comment. The syphilis case was decided as indicated on the syphilis screening algorithm included as Figure 1.b. Yes, titres were measured for VDRL for the majority of the sentinel sites to determine if it was a current infection or an old/scarring infection, but unfortunately this information was not collected during the survey. We also add this as a limitation in the discussion. The algorithm was traditional.

• Lines 130- Is the CSPRO software a registered brand? Add developer, city and country

Authors: We have added developer, city and country, Thank you.

• Line 138 – the authors refer to a sub-set to be tested for syphilis. How was this subset selected. Later on they state that shortage of test kits meant sites with no test kits could not be selected for the analysis.

Authors: Thank you to reviewer for this comment. The subset of analysis was selected based on the available syphilis test results. In fact, all sites performed syphilis testing, but the stockout experienced by some sites at one point during the study unfortunately led to some study participants not being tested and were excluded from the analysis.

Results

• Line 163 – please change from involvement to particioation

Authors: We changed involvement to participation, Thank you.

• Table 1- present medians (IQR) as well as age in cvategories

Authors: We have prevented medians (IQR) for age and we have commented, Thank you.

• Table 2 – present numbers before percentages

Authors: We have presented numbers before percentages Thank you.

• Table 3: for overall population and those HIV positives add the values of N- the totals. Also primiparous and number of pregnancies overlap as variables. Primiparous and # pregnancies =0 are one and the same thing.

Authors: Thank you for this interesting comment. The totals for the overall population and the HIV-positive were added. For the variables Primiparous and number of pregnancies, they were kept in the analysis to test in parallel the effect of first pregnancy on the risk of Syphilis and HIV infection and to see if a particular effect was found in the heterogeneous group of multiparous women in the risk of Syphilis and HIV infection via the variable number of pregnancies.

• Table 4: see comment above

Authors: Thank you for this pertinent comment. For the variables Primiparous and number of pregnancies, they were kept in the analysis to test in parallel the effect of first pregnancy on the risk of Syphilis and HIV infection and to see if a particular effect was found in the heterogeneous group of multiparous women via the variable number of pregnancies. Because these variables overlap, we considered only the primiparous variable in our multivariate analysis.

• Fig 1: add syphilis testing algorithm

Authors: The Syphilis algorithm have been added as Figure 1.b and Fig 1 is now Fig 1.a, Thank you.

Discussion

• Lines 278- 286: the authors have not addressed the issue of access to treatment as a factor that could explain high rates of syphilis in rural areas. From about 2016, there has been a global benzathine penicillin shortage. Please comment on this

Authors: Thank you to reviewer 1 for this interesting comment. As recommended in the WHO guide, syphilis (new or old infection) is cure in first intention with benzathine penicillin. WHO also recommended when benzathine or procaine penicillin cannot be used (e.g. due to penicillin allergy where penicillin desensitization is not possible) or are not available (e.g. due to stockouts), the WHO STI guideline suggests using, with caution, erythromycin 500 mg orally four times daily for 30 days [1]. Although erythromycin treats the pregnant women, it does not cross the placental barrier completely and as a result the fetus is not treated. It is therefore necessary to treat the newborn infant soon after delivery (see recommendations 9 and 10 in the WHO guidelines for the treatment of syphilis, which refer to congenital syphilis). Doxycycline should not be used in pregnant women. Because syphilis during pregnancy can lead to severe adverse complications to the fetus or newborn, stockouts of benzathine penicillin for use in antenatal care should be avoided [1].

Thus the global stockout of benzathine penicillin in 2016 could have play a role in the sero-prevalence rate and the spread of syphilis, particularly in rural areas[2]. We added this in the discussion (lines 291- 294).

Reference: [1] « WHO | WHO guideline on syphilis screening and treatment for pregnant women », WHO. http://www.who.int/reproductivehealth/publications/rtis/syphilis-ANC-screenandtreat-guidelines/en/ (consulté le oct. 11, 2020).

[2] S. Nurse-Findlay et al., « Shortages of benzathine penicillin for prevention of mother-to-child transmission of syphilis: An evaluation from multi-country surveys and stakeholder interviews », PLoS Med, vol. 14, no 12, déc. 2017, doi: 10.1371/journal.pmed.1002473.

Lines 296- 308: The list of limitations is incomplete. Because selection of sites for syphilis testing were not random, discuss that it was possible that sites which higher syphilis rates could have been included in the survey

Authors: Thank you to reviewer for this comment. We had included this limitation in the actual version of the manuscript (line 291 – 294).

26 Oct 2020

Highlighting a population-based re-emergence of Syphilis infection and assessing associated risk factors among pregnant women in Cameroon: Evidence from the 2009, 2012 and 2017 national sentinel surveillance surveys of HIV and syphilis.

PONE-D-20-11194R2

Dear Dr. KENGNE-NDE,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Remco PH Peters, MD, PhD, DLSHTM

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: