Cardiovascular disease risk factors and markers of oxidative stress and DNA damage in leprosy patients in Southern Nigeria.

Leprosy reduces quality of life of affected persons. Oxidative stress caused by reactive oxygen species may play a vital role in the pathogenesis of leprosy. This study evaluated anthropometric indices, fasting plasma glucose (FPG), lipid profile, total antioxidant capacity (TAC), total plasma peroxide (TPP), oxidative stress index (OSI), malondialdehyde (MDA), glutathione (GSH) and 8-hydroxy-2-deoxyguanosine (8-OHdg) in leprosy patients. Sixty test participants of both genders, aged 18-65years and diagnosed of multibacillary leprosy and 30 apparently healthy controls were consecutively recruited for this study. The test participants comprised of 30 patients on multidrug therapy (MDT) and 30 patients relieved from therapy (RFT). Body mass index (BMI), Waist-hip ratio (WHR), FPG, lipid profile, TAC, TPP, OSI, MDA, GSH and 8-OHdg were determined using appropriate methods. Data were analyzed using Analysis of variance; p<0.05 was considered statistically significant. The MDT group had significantly lower BMI (p = 0.0001), Total cholesterol (p = 0.001), HDL-C (p = 0.019), LDL-C (p = 0.005), TAC (p = 0.0001) and higher TPP (p = 0.001), MDA (p = 0.0001), OSI (p = 0.005) and 8-OHdg (p = 0.035) compared to the controls. The RFT group had significantly lower BMI (p = 0.001) Total cholesterol (0.0001), HDL-C (p = 0.006) LDL-C (p = 0.0001), TAC (p = 0.001) and higher WHR (p = 0.010), VLDL-C (p = 0.035), TG (p = 0.023) Atherogenic index of plasma (p = 0.0001) and TPP (p = 0.001), MDA (p = 0.0001) compared to the control group. GSH levels correlated negatively with duration of treatment (r = -0.401, p = 0.028). This study has shown that there is oxidative stress in multibacillary leprosy patients irrespective of drug treatment status. This study also shows that leprosy patients relieved from treatment may be susceptible to cardiovascular events. Antioxidants supplementation may be beneficial in the treatment of leprosy and clinical follow up on patients relieved from treatment may also be necessary to monitor health status and prevent development of cardiovascular events.

Introduction

Leprosy or Hansen’s disease is a chronic debilitating disease caused by Mycobacterium leprae that affects the skin, peripheral nerves, mucosa of the upper respiratory tract, and the eyes causing progressive and permanent disabilities if not treated [1]. Although Leprosy was eliminated as a public health problem (i.e. a disease burden of < 1 case per 10 000 persons) globally in the year 2000, leprosy still remains a global public health issue [2]. Each year about 200–300 thousand people are diagnosed of leprosy and about 2–3 million people are disabled because of it [3]. Official figures from 150 countries from 6 WHO regions show that globally 7, 607, 837 persons are infected with leprosy [4]. A global registered prevalence of 192, 713 cases was reported in 2017, an increase by 20, 765 cases over that in 2016, and 210, 671 new cases were detected in the same year [5].

In Nigeria a registered prevalence of 3,234 cases was reported in 2015 and 2,892 new cases were detected in the same year [6]. With this, Nigeria ranked third among African countries with the highest burden of the disease [7]. However in 2017, prevalence of 11, 230 cases was reported and 2,447 new cases detected, with a total of 195, 875 persons infected with leprosy in the country; making Nigeria first among African countries with the highest burden of the disease in 2017 [5]. Nigeria Center for Disease Control (NCDC) moreover maintains that over 3,500 people are diagnosed with leprosy yearly in the country with about 25% of victims having some degree of disability [8].

The pathogenesis of this neurodegenerative disease is not fully understood [9]. These leprosy-associated disabilities potentially caused by damage to the peripheral nerves have been linked to a number factors including oxidative stress [10]. Excessive production of reactive oxygen species (ROS) is the major precursor of Oxidative Stress (OS). Oxidative Stress is a condition associated with an increased rate of metabolic impairment and cellular damage occurring due to derangement in the balance between ROS and the body’s antioxidant system [11]. It is suggested that the possible reason for decreased antioxidant status in leprosy cases may be increased production of ROS via cell mediated immunity, as well as the free radical producing ability of drugs used in multidrug therapy (MDT) of leprosy [12]. Of the various mechanisms that influence the pathogenesis of leprosy, oxidative stress may well be important; however, only a few studies have been done to assess the status of oxidative stress in leprosy patients via markers of oxidative stress such as Glutathione (GSH), Superoxide dismutase (SOD), and lipid peroxidase product, Malondialdehyde (MDA). 8-hydroxy-2deoxy guanosine (8-OHdG) is a DNA nucleoside which is an oxidative derivative of guanosine [10]. Measurement of the levels of 8-OHdG is important as a biomarker of oxidative stress causing cellular DNA damage. Increased levels of 8-OHdG is associated with a lot of pathologic conditions like cancer, diabetes, and hypertension [13].

Cardiovascular diseases (CVD) are the leading cause of death and disability worldwide. Together they resulted in 17.9 million deaths in 2015 up from 12.3 million in 1990 and represented 31% of all global deaths in 2016 [4]. Growing evidence indicates that chronic and acute over production of reactive oxygen species under pathophysiologic conditions is integral in the development of cardiovascular diseases [14]. Majority of cardiovascular disease results from complications of atherosclerosis, and an important initiating event for atherosclerosis may well be the oxidation of low-density lipoprotein (LDL) and the transport of oxidized low-density lipoprotein (Ox-LDL) across the endothelium into the artery wall [15]. Dyslipidaemia, hyperglycaemia, and insufficient physical activity are cardiovascular risk factors that have been associated with leprosy [16, 17].

In spite of the global interest in CVD and the growing interest in oxidative stress and its role in the aetiology of many diseases, very few studies on these have been conducted among people affected by leprosy. The few studies evaluating oxidative status in leprosy patients were conducted in India and Brazil [11, 12, 16, 18] and none amongst African people affected with leprosy. There is also dearth of information regarding the link between leprosy and the risk of cardiovascular disease in Africans, hence the need for this study. The purpose of this study was to assess the cardiovasculardisease risk factors and markers of oxidative stress and DNA damage in a native black population of leprosy patients to see if there are any deleterious changes on these markers especially in those undergoing multiple drug therapy and those relieved from treatment. The study assessed anthropometric indices, fasting plasma glucose, lipid profile, total antioxidant capacity (TAC), total plasma peroxide (TPP), oxidative stress index (OSI), malondialdehyde (MDA), glutathione (GSH) and 8-hydroxy-2-deoxyguanosine (8-OHdg) in patients with leprosy.

Methodology

Ethics statement

This study was carried out in accordance with the World Medical Association’s Declaration of Helsinki [19]. Ethical clearance was obtained from Health Research and Ethics Committee of the Akwa Ibom State Ministry of Health, Nigeria (Ref No.: MH/PRS/99/vol.IV/452). The study participants were informed of the nature of the research and written informed consent obtained as approved by the ethics committee before they were enrolled for the study.

Study design and subject selection

A case-control study design was used for the study and was carried out in Ekpene Ebom, Etinan LGA, in Akwa Ibom State Nigeria. A total of 90 (male and female) participants were consecutively recruited for the study. They consisted of 60 known multibacillary leprosy patients as test subjects and 30 apparently healthy individuals who did not have leprosy as controls. They were aged between 18 to 65 years and were all Nigerians. The leprosy patients were recruited from outpatients and inpatients of the Leprosy Referral Hospital in Ekpene Ebom, and 30 apparently healthy individuals were recruited as control participants from Akwa Ibom State. The 60 test participants comprised of 30 patients on multidrug therapy (MDT) and 30 patients who have been relieved from treatment (RFT). Treatment modality for the patients was the administration of Dapsone 100 mg /daily, Rifampicin 600 mg/daily and Clofazimine 50 mg /daily for 24 months. The study was carried out from 1 September 2018 to 1 August 2019. A standard questionnaire was administered and sociodemographic data obtained.

Measurement of anthropometric indices and blood pressure and definition of cut-off

Each participant’s weight (in kilograms) and height (in metres) were measured. A weighing balance was utilized to measure weight in kilograms and a stadiometer was used to measure height in meters. Body mass index (BMI) was calculated by as the ratio of the weight to the square of height (kg/m2). Normal range for BMI is 18–25 kg/m2. Obesity was defined as BMI ≥30 kg/m2. Waist circumference (in centimeters) was obtained by taking two measurements, one after inhalation and the other after exhalation at the midpoint between the top of the iliac crest and the bottom of the rib cage using an inelastic calibrated tape. Hip circumference measured at the hips and buttocks. Waist-to-hip ratio was defined as waist girth/hip circumference. Obesity was defined as Waist/hip ratio >0.9 (men), >0.85 (women). The blood pressure was measured on the right arm with a mercury sphygmomanometer (cuff size 12.5 X 40 cm) with the patient in a seated position and after a 5-minute rest. The systolic and diastolic blood pressures were recorded. Hypertension was defined as blood pressure ≥140/90 mmHg or the use of antihypertensive medications [20].

Inclusion and exclusion criteria

The test participants were recruited not withstanding impairment/disability status. Leprosy patients who had just been diagnosed and had not started any treatment regimen, those refused their consent to participate and non-infected persons with any sign or symptoms of illness at the time of the study were excluded.

Collection of sample

Seven millilitres (7ml) whole blood was collected aseptically by venepunture using a Terumo vacutainer (Terumo, Japan) from each participant after an overnight fast. Two millilitres (2ml) was dispensed into fluoride oxalate bottles for fasting plasma glucose estimation while five millilitres (5ml) was dispensed into a plain bottle, allowed to clot at room temperature, centrifuged at 3000rpm for 5minutes and serum extracted and and stored at -20°C.

Estimation of analytes

Fasting plasma glucose (FPG) and lipid profile

Fasting plasma glucose (FPG) was estimated by the glucose oxidase method of Trinder, [21]. Total cholesterol (TC) was estimated by the method of Allain et al., [22] and Triglycerides by the method of Bucolo and David, [23]. Estimation of high density lipoprotein cholesterol (HDL-C) was by Precipitation and cholesterol determination method of Demacker et al., [24] and Allain et al., [22] respectively. All the kits were obtained from Randox (Antrim, United Kingdom). Low Density Lipoprotein Cholesterol (LDL-C) was calculated using Friedewald’s equation: LDL-C = TC—HDL-C—VLDL-C [25] while Very Low Density Lipoprotein Cholesterol (VLDL-C) was calculated using the equation: VLDL-C (mmol) = TG/2.2 provided TG was ≤ 4.5mmol/l [26]. Normal ranges for the variables were as follows: FPG— 3.5—5.5mmol/L; TC: 3.0—6.5mmol/L; HDL-C: 0.9—1.5mmol/L; LDL-C: 2.5—3.8mmol/L; VLDL-C: 0.4—0.6mmol/L; TG: 1.2mmol/L. Atherogenic Index Plasma was calculated according to the formula, log (TG/HDL-C) [27]. Subjects with AIP value of > 0.11 were considered at elevated cardiovascular risk.

Oxidative stress markers

Determination of glutathione (GSH) was based on the methodology described by Ellman, [28] and malondialdehyde (MDA) was by the method of Buege and Aust [29]. Total antioxidant capacity was determined by the method of Koracevic et al., [30]. Total plasma peroxide (TPP) was determined using the reaction of ferrous-butylated hydroxytoluene-xylenol orange complex ‘FOX2’ method of Miyazawa, [31] with minor modifications as described by Harma et al., [32]. The ratio of total plasma peroxide (TPP) to total antioxidant capacity (TAC) was calculated as the oxidative stress index, an indicator of the degree of oxidative stress.

Marker of oxidative DNA damage

Estimation of 8-OHdG was done using Agisera’s ELISA kit no: AS152887 (Agrisera, Vännäs, Sweden). The Agrisera’s 8-OHdG ELISA is a competitive assay for the quantitative measurement of 8-OHdG. It utilizes an 8-OHdG coated plate and an HRP-conjugated antibody for detection which allows for an assay range of 0.94-60ng/L, with a sensitivity of 0.59ng/L. The 8-OHdG antibody used in this assay recognizes both the free 8-OHdG and the DNA incorporated 8-OHdG. All the analytes were estimated in the Chemical Pathology Laboratory of the University of Calabar Teaching Hospital, Calabar, Nigeria.

Statistical analysis

Data was analyzed using the PAWstatistic 18, a statistical package from SPSS Inc, (Chicago IL, United States of America) and R version 4.0.0 from The R Foundation for Statistical Computing Platform, Vienna, Austria. Results were presented as mean ± standard deviation. Chi-squared test of independence was to test significant relationship between categorical variables. The assessment of the normality of the data was done using the Shapiro-Wilk’s test. Comparisons of groups were made using Students’ t-test and analysis of variance (ANOVA) and post hoc analysis using Least significant difference. Pearson’s correlation analysis was also done. The level of significance was set at 95% confidence interval, where p-value less than 0.05 (p<0.05) was considered as statistically significant. Graphs were created with Microsoft excel 2007 version (Microsoft Corporation, US).

Results

Table 1 shows the sociodemographic data of leprosy patients on MDT, those RFT and controls. The groups have similar sex distribution, but those relieved from treatment are older than both the controls and those undergoing treatment. The occupation of the leprosy patients was mostly farming and petty trading. However, a large proportion (60%) of those relieved from treatment were unemployed. In the MDT group, a significantly large proportion (83.3%), compiled strictly to treatment regimen with only 16.7% (n = 5) lagging in compliance. There was no co-infection with HIV in the test group. The leprosy patients relived from treatment had a significantly higher (p = 0.0001) frequency of physical deformity as well as history of leprae reactions.

Table 1

Sociodemographic data of leprosy patients undergoing multidrug therapy, leprosy patients relieved from treatment and control.

Variable	Leprosy patients undergoing multidrug therapyn = 30	Leprosy patients relieved from treatmentn = 30	Controlsn = 30	P-value
Age	38.4± 16.80	49.8± 9.76#	38.0± 12.79	0.001
Occupation	Students 3 (10%)Traders 11(36.7%)Farmers 16 (53.3%)	Farmers 6(20%)Traders 6(20%)Unemployed 18(60%)	Students 7 (23.3%)Farmers 5(16.7%)Traders 5(16.7%)Civil servants 13(43.3%)
Gender	Male 19 (63.3%)Female 11 (36.7%)	Male 22 (73.3%)Female 8 (26.7%)	Male 17 (56.7%)Female 13 (43.3%)	0.398
Compliance with treatment	Compliant: 25 (85%)*Not Complaint: 5 (15%)
Physical deformity	4 (13.3%)	20(66.7%)	Nil	0.0001
History of Leprae Reactions	3(10%)	21(70%).	Nil	0.0001
Duration of MDT (months)	7.3±6.11(Min (0.5); Max(23)
Duration relieved from treatment(yr)		3.5±1.96(Min (1.0); Max(8.0)

Table 2 shows the comparison of anthropometric indices, blood pressures, lipid profile, atherogenic index of plasma and oxidative stress markers in leprosy patients undergoing multiple drug therapy, leprosy patients relieved from treatment and controls. The result shows that there was a significant variation in the levels of BMI (p = 0.0001), WHR (p = 0.033), DBP (p = 0.048), TC (p = 0.0001), HDL-C (p = 0.013), LDL-C (p = 0.001), VLDL-C (p = 0.047), TG (p = 0.037), AIP (p = 0.001), TAC (p = 0.0001), TPP (p = 0.001), OSI (p = 0.018), MDA (p = 0.0001), GSH (p = 0.036) and 8-OHdG (p = 0.015) among the groups. There was no significant variation (p>0.05) in the SBP and FPG levels among the groups. Due to the fact that the RFT were older than both controls and MDT group we adjusted for age by adding age as a covariate into the analysis and the results did not change.

Table 2

Anthropometric indices, blood pressures, lipid profile, atherogenic index of plasma and oxidative stress markers in leprosy patients undergoing multiple drug therapy (MDT), leprosy patients relieved from treatment (RFT) and controls.

Parameter	Leprosy Patients		Controls n = 30	p-value
	MDTn = 30	RFTn = 30
BMI (kg/m2)	19.8 ± 4.30	20.3 ± 3.94	23.6± 2.58	0.0001*
Waist/Hip ratio	0.86 ±0.04	0.89 ±0.08	0.85 ±0.05	0.033*
Systolic BP (mmHg)	113.3 ±14.16	122.7 ±22.73	116.8 ±6.58	0.078
Diastolic BP (mmHg)	71.8 ±7.93	74.0 ±11.91	78.2 ±9.74	0.048*
FPG (mmol/L)	6.19 ± 7.25	4.38 ± 0.64	4.42 ± 0.47	0.173
Total cholesterol (mmol/L)	3.92 ± 0.86	3.46 ± 0.97	4.68 ± 0.71	0.0001*
HDL-cholesterol (mmol/L)	1.41 ± 0.39	1.37 ± 0.35	1.65 ± 0.41	0.013*
LDL-cholesterol (mmol/L)	2.05 ± 0.69	1.59 ± 0.92	2.61 ± 0.58	0.0001*
VLDL-cholesterol (mmol/L)	0.45 ± 0.25	0.56 ± 0.20	0.45 ± 0.14	0.047*
Triglycerides (mmol/L)	0.99 ± 0.54	1.24 ± 0.43	0.98 ± 0.30	0.037*
Atherogenic index of plasma	-0.18 ±0.20	-0.05 ±0.19	-0.23 ±0.18	0.001*
TAC (mmol)	1.20 ± 0.45	1.40 ± 0.50	1.94 ± 0.76	0.0001*
TPP (μmol/L)	111.8 ± 11.66	112.5 ± 8.97	104.2 ± 6.55	0.001*
Oxidative Stress index	12.0 ± 9.07	9.77 ±5.59	6.9 ±4.75	0.018*
MDA (mmol/L)	0.74 ± 0.18	0.87 ± 0.23	0.52 ± 0.10	0.0001*
GSH (mg/dL)	79.1 ± 20.34	79.2 ± 20.67	90.6 ± 29.70	0.111
8-OHdg (ng/L)	9.2 ± 7.98	5.67 ± 1.36	6.44 ± 1.80	0.015*

Results expressed as Mean ± SD

*significant at p<0.05

A post hoc analysis showed that the mean values of BMI (p = 0.0001), DBP (p = 0.016), TC (p = 0.001), HDL-C (p = 0.019) and LDL-C (p = 0.005) and TAC (p = 0.0001) were significantly lower while the mean values of TPP (p = 0.001), OSI (p = 0.005), MDA (p = 0.0001) and 8-OHdg (p = 0.035) were significantly higher in the MDT group when compared to controls. The mean values of BMI (p = 0.001), TC (p = 0.0001), HDL- C (p = 0.006), LDL-C (p = 0.0001) and TAC (p = 0.001) were significantly lower in the RFT group while the mean values of WHR (p = 0.010), VLDL-C (p = 0.035), TG (p = 0.023), AIP (p = 0.0001), TPP (p = 0.001) and MDA (p = 0.0001) were significantly higher in the RFT group when compared to the controls. Further comparison between the MDT and RFT group showed that TC(p = 0.041), LDL-C(p = 0.019) and 8-OHdg (0.006) levels were significantly lower while VLDL-C (p = 0.050), TG(p = 0.030), AIP (p = 0.011), MDA (p = 0.004) and were significantly higher in the RFT group when compared to the MDT group (Table 3).

Table 3

Comparison of anthropometric indices, blood pressures, lipid profile, atherogenic index of plasma and oxidative stress markers in leprosy patients undergoing multiple drug therapy (MDT), leprosy patients relieved from treatment (RFT) and controls using post hoc analysis.

Parameter	Groups		Mean Difference	Std Error	p-value
	MDTn = 30	Controls n = 30
BMI (kg/m2)	19.8 ± 4.30	23.6± 2.58	-3.800	0.951	0.0001
Diastolic BP (mmHg)	71.8 ±7.93	78.2 ±9.74	-6.367	2.581	0.016
Total cholesterol (mmol/L)	3.92 ± 0.86	4.68 ± 0.71	-0.760	0.221	0.001
HDL-cholesterol (mmol/L)	1.41 ± 0.39	1.65 ± 0.41	-0.237	0.099	0.019
LDL-cholesterol (mmol/L)	2.05 ± 0.69	2.61 ± 0.58	-0.560	0.192	0.005
TAC (mmol)	1.20 ± 0.45	1.94 ± 0.76	0.733	0.151	0.0001
TPP (μmol/L)	111.8 ± 11.66	104.2 ± 6.55	7.643	2.401	0.001
Oxidative Stress index	12.0 ± 9.07	6.9 ±4.75	5.047	1.739	0.005
MDA (mmol/L)	0.74 ± 0.18	0.52 ± 0.10	0.210	0.046	0.0001
8-OHdg (ng/L)	9.2 ± 7.98	6.44 ± 1.80	2.772	1.290	0.035
	RFTn = 30	Controlsn = 30
BMI (kg/m2)	20.3 ± 3.94	23.6± 2.58	-3.343	0.951	0.001
Waist/Hip ratio	0.89 ±0.08	0.85 ±0.05	0.041	0.015	0.010
Total cholesterol (mmol/L)	3.46 ± 0.97	4.68 ± 0.71	-1.217	0.221	0.0001
HDL-cholesterol (mmol/L)	1.37 ± 0.35	1.65 ± 0.41	-0.277	0.099	0.006
LDL-cholesterol (mmol/L)	1.59 ± 0.92	2.61 ± 0.58	-1.020	0.192	0.0001
VLDL-cholesterol (mmol/L)	0.56 ± 0.20	0.45 ± 0.14	0.111	0.052	0.035
Triglycerides (mmol/L)	1.24 ± 0.43	0.98 ± 0.30	0.260	0.112	0.023
Atherogenic Index of plasma	-0.05 ±0.19	-0.23 ±0.18	0.182	0.049	0.0001
TAC (mmol)	1.40 ± 0.50	1.94 ± 0.76	-0.540	0.151	0.001
TPP (μmol/L)	112.5 ± 8.97	104.2 ± 6.55	8.330	2.401	0.001
MDA (mmol/L)	0.87 ± 0.23	0.52 ± 0.10	0.347	0.046	0.0001
	MDTn = 30	RFTn = 30
Total cholesterol (mmol/L)	3.92 ± 0.86	3.46 ± 0.97	-0.457	0.221	0.041
LDL-cholesterol (mmol/L)	2.05 ± 0.69	1.59 ± 0.92	0.460	0.192	0.019
VLDL-cholesterol (mmol/L)	0.45 ± 0.25	0.56 ± 0.20	-0.102	0.052	0.050
Triglycerides (mmol/L)	0.99 ± 0.54	1.24 ± 0.43	-0.247	0.112	0.030
Atherogenic Index of plasma	-0.18 ±0.20	-0.05 ±0.19	-0.128	0.049	0.011
MDA (mmol/L)	0.74 ± 0.18	0.87 ± 0.23	-0.137	0.046	0.004
8-OHdg (ng/L)	9.2 ± 7.98	5.67 ± 1.36	3.550	1.256	0.006

Table 4 shows the correlation of duration of treatment and some other indices in leprosy patients undergoing multidrug therapy. Results showed a significant positive correlation between duration of treatment and TC (r = 0.611, p = 0.0001), HDL-C (r = 0.364, p = 0.048), LDL-C (r = 0.416, p = 0.022) and TG (r = 0.364, p = 0.048). However, a negative significant correlation was observed between duration of treatment and GSH (r = -0.401, p = 0.028).

Table 4

Correlation of duration of treatment and some other indices in leprosy patients undergoing multiple drug therapy.

Parameter	Index	r-value	p-value
Duration of treatment (months)	Body mass index	0.272	0.146
	Systolic BP	0.152	0.422
	Diastolic BP	-0.297	0.111
	Fasting plasma glucose	-0.077	0.687
	Total cholesterol	0.611	0.0001*
	HDL-cholesterol	0.364	0.048*
	LDL-cholesterol	0.416	0.022*
	VLDL-cholesterol	0.342	0.064
	Triglycerides	0.364	0.048*
	Atherogenic Index of plasma	0.166	0.379
	Total antioxidant capacity	0.230	0.221
	Total plasma peroxide	-0.331	0.074
	Oxidative Stress index	-0.260	0.165
	Malondialdehyde	-0.272	0.147
	Glutathione	-0.401	0.028*
	8-OHdg	0.148	0.148

*Significant at p<0.05

Discussion

The study assessed fasting plasma glucose, lipid profile, total antioxidant capacity, total plasma peroxide, oxidative stress index, malondialdehyde, glutathione and 8-OHdg in patients with leprosy on mutltidrug therapy (MDT) and those relieved from therapy (RFT). The WHO health Organization has assiduously worked towards a leprosy-free world through its Global Leprosy Strategy 2016–2020 [33] and so has the National Tuberculosis and Leprosy Control Programme in Nigeria. However, more has to be done towards reintegrating those who have been cured of the disease into society. From our study, 60% of the leprosy patients that were relieved from treatment were unemployed. More than two thirds of the patients were males who should supposedly be breadwinners for their families.

Significantly lower levels of total cholesterol (TC), HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) were observed in patients on MDT and those RFT compared to the controls and significantly higher levels of VLDL-cholesterol (VLDL-C), triglycerides (TG) and Atherogenic index of plasma (AIP) in patients RFT compared to the controls. The low TC levels obtained in this study agrees with the findings of Sheikh et al., [34] and Nega et al., [35] who also detected lower TC levels in multibacillary form of the disease. However Silva et al., [16] reported normal levels of serum TC in both paucibacilliary and multibacilliary forms of the disease and other authors have even reported an increase in serum TC in patients with lepromatous leprosy [36]. The low serum TC observed in this study may be due to increase utilization of cholesterol by M. leprae for nutrition and persistence of the bacilli [18] or due to hepatic involvement as evidenced by several studies indicating hepatic damage by M. leprae bacilli [36]. It is also suggested that in lepromatous leprosy, free fatty acids are increased due to microbial modulation of lipoprotein lipase activity, which exerts an inhibitory effect on hepatic lipogenesis directly and indirectly on cholesterol synthesis by inhibiting pyruvate dehydrogenase enzymes of the liver [37].

The leprosy patients in both groups (MDT and RFT) had significantly lower HDL-C levels compared to the controls. This result differs from the findings of Silva et al., [16] and Sheikh et al. [34] who reported normal and significantly higher serum HDL-C levels in leprosy patients respectively but corroborate the findings of Gupta et al., [37] and Nega et al., [35] who detected significantly lower HDL-C levels in patients with the lepromatous form of the disease compared to the controls. It is suggested that this low serum HDL-C level observed could be due to increase in the levels of cytokines such as tumor necrosis factor (TNF-α) which lowers the HDL levels [35, 37]. Pro- inflammatory immune response against microbial infections up regulates the production of cytokines like TNF -α, IL-1, IL-2 & IL-6 as a host protective mechanism against bacterial assault [38]. TNF-α is a critical host-protective cytokine against mycobacterial diseases that decreases HDL levels [39] and studies have shown that TNF-α levels are increased in PB lesions and episodes of reverse reaction [40,41]. The precise mechanism by which TNF-α decrease HDL levels is uncertain and is likely to involve multiple mechanisms. It decreases the production of apo-A1, enhances HDL degradation by macrophages, decreases Lecithin Cholesterol Acyltransferase (LCAT) activity, causes an increase in serum amyloid A production, induces secretory phospholipase A2 (sPLA2) and endothelial cell lipase (HDL-metabolizing enzymes) which may all result in decreased HDL levels [38, 42].

Significantly lower levels of LDL-C were observed in multibacillary leprosy patients irrespective of drug therapy status when compared to the controls in this study. This agrees with the findings of a study Sheikh et al., [34]. Normal LDL-C levels were however observed by Silva et al. [16] in MB leprosy patients when compared to controls. In the present study, serum LDL-C level was also shown to be significantly lower in participants relieved from therapy when compared to those on MDT. The low LDL-C levels observed in this study may be due to impact of infection on hepatic lipogenesis. Nwosu & Nwosu [36] observed significantly high levels of alanine amino transferase in MB leprosy and Dhavalshankh et al., [43] observed significantly lower levels of albumin in lepromatous leprosy and these studies concluded that leprosy is associated with liver damage. The liver is the principal organ involved in the lipid metabolism and has a major role in the control of plasma LDL-C level since most LDL-C receptors are present in the liver; therefore its invasion by leprae bacilli may alter lipid metabolism [44].

This study showed significantly higher levels of VLDL and TG in MB leprosy patients relieved from treatment when compared to controls. This is similar to the work of Nega et al., [35]. However, normal triglyceride levels in leprosy patients with no significant variation in VLDL levels have been reported by other authors [16, 45]. The higher levels of triglyceride and VLDL-C cholesterol may be attributed to several mechanisms, including reduction of TG hydrolysis, lipopolysaccharide- and pro-inflammatory cytokine-induced de novo free fatty acid production and TG synthesis in the liver as well as reduction of lipoprotein lipase activity thus resulting in reduced VLDL clearance and increased TG levels [38]. The decreased clearance of TG-rich lipoproteins may be due to continued induction of cell-mediated inflammatory response caused by the presence of M. leprae antigens even after completion of therapy [46]. Some cytokines (TNF-α, IL-1, IL-2, IL-6) have been shown to decrease the synthesis of lipoprotein lipase, the key enzyme that metabolizes triglycerides in circulation, and inflammatory responses increases the production of these cytokines. Studies have also shown that inflammation also increases angiopoietin like protein 4, an inhibitor of lipoprotein lipase activity, which would further block the metabolism of VLDL and TG [47]. It may also be important to note that the increase in may also be due to age–related factors since those relieved from treatment were older than the controls, however this effect is mild as the statistical differences observed still remained after adjusting for age.

This work also showed positive significant correlation in TC, HDL-C and LDL-C levels with duration of treatment. This agrees with the observation that MDT may benefit hepatic lipogenesis and plasma lipid and lipoprotein abnormalities/levels may even return towards normal following recovery from the infection [42]. This study also showed significant increase in TG levels following increase in treatment duration. Some anti-inflammatory drugs have effects on lipid metabolism that are independent of the reduction in inflammation. For example, high dose glucocorticoid treatment results in an increase in serum triglyceride and VLDL levels due to the increased production and secretion of VLDL by the liver [42]. Because all MDT regimens for leprosy contain Clofazimine, which has strong anti-inflammatory activity [48], MDT may therefore increase VLDL secretion in the liver leading to increase in plasma TG levels.

Atherogenic index of plasma is a novel indicator of dyslipidemia and associated cardiovascular disease [49]. This study showed a significantly higher AIP in multibacillary leprosy patients when compared to controls. Inflammation and infections induce a variety of alterations in lipid metabolism that if prolonged could contribute to the increased risk of atherosclerosis. The most common changes are decreases in serum HDL-C and increases in triglyceride levels. In addition to affecting serum lipid levels, inflammation also adversely affects lipoprotein function. LDL is more easily oxidized as the ability of HDL to prevent the oxidation of LDL is diminished leading to atherosclerosis [42]. AIP was also significantly higher in the RFT group when compared to patients on MDT. This agrees with recent findings that cytokines mediated inflammatory responses, perhaps to persistent M. leprae antigen, still occur in leprosy patients even after being declared cured [35, 46]. Inflammation reduces serum HDL-C and increases TG levels and could contribute to the increased risk of atherosclerosis [42].

Results from this study also showed that the BMI of the both groups of leprosy patients compared to the controls was significantly lower indicating wasting, maybe due to malnutrition [12]. However, the WHR of leprosy patients relieved from treatment was significantly higher when compared to the control group. This agrees with the work of Silva et al., [16] who observed a higher waist circumference in multibacillary leprosy patients compared to controls. This suggests that WHR may be a better indicator of adiposity in these patients and leprosy.

The observed higher levels of TG, VLDL-C, AIP, WHR and lower levels of HDL-C observed in patients RFT compared to controls, suggests that leprosy patients who are relieved from treatment may be predisposed to developing a cardiovascular disease. This may result in a double burden of disease as well as increased mortality for those who suffer from this disease.

In this study oxidative stress markers assessed included total antioxidant capacity (TAC), total plasma peroxide (TPP), oxidative stress index (OSI), malondialdehyde (MDA), glutathione (GSH) while 8-hydroxy-2-deoxyguanosine (8-OHdg) was assessed as a marker of oxidative DNA marker. The significantly lower levels of TAC and GSH and higher levels of TPP, MDA in both groups of patients (MDT and RFT) and higher OSI and 8-OHdg in only the MDT group when compared to the controls suggests the presence of increased oxidative stress. These findings agree with the works of Prabhakar et al., [11] who showed that there is oxidative stress in MB leprosy cases, irrespective of their treatment status. Oxidative stress has been suggested to play a vital role in the pathogenesis of leprosy [12]. The leprosy patients exhibit increased oxidative stress which could mediate inflammatory episodes, depressed cell-mediated immune response, organ damage as well as degeneration of nerves in these patients [12]. Oxidative stress not only occurs in newly diagnosed cases but also in patients who have finished the therapy [50]. Higher MDA levels observed in RFT group suggests that there may be continued induction of ROS by persistent M. leprae antigen or remnant bacilli even after completion of therapy [46]. Significantly higher MDA levels indicate that increased lipid peroxidation and DNA damage due to free radical mediated injury occurs in leprosy patients [51]. A major defence against microbial infection is the macrophage system [52]. The macrophages infected by mycobacteria show increased phagocytosis, enzyme activity and oxygen consumption known as “respiratory burst,” [12]. Microbial killing by macrophages is associated with a burst of respiratory activity that also leads to production of ROS [51]. Another probable factor that may encourage lipid peroxidation in leprosy patients may the oxidative stress that is associated with amyloid formation and deposition [52]. Amyloid deposits have been reported in some leprosy patients with prevalence from autopsy studies ranging from 3.9% and 31% [53]. Major targets of ROS-associated peroxidation are the polyunsaturated fatty acids (PUFA) found in membrane lipids. PUFA is degraded by free radicals to form malondialdehyde (MDA) therefore MDA in serum serves as a marker of cellular damage due to free radicals [12]] and high serum MDA levels is indicative of increased lipid peroxidation and DNA damage by free radicals [54].

Antioxidant status is an index of oxidative stress. High level of oxidative stress is also shown by the low cellular antioxidant status [50] and decrease in antioxidant defense may be one of the reasons for increased levels of ROS and subsequent tissue damage in lepromatous spectrum [55]. In this study, both the RFT and MDT leprosy patients had lower TAC levels compared to controls although there was no significant variation in their GSH levels. Antioxidants which play a central role in maintaining the cells redox state both enzymatically and non-enzymatically by neutralizing free radicals, notably superoxide radicals, hydroxyl radicals, nitric oxide and carbon [56]. Studies have also shown that GSH is considered a potent inhibitor of lipid peroxidation process, and thus regulates the MDA content [57]. The comparable GSH levels in the leprosy patients compared with the controls contradicts findings in studies by Prabhakar et al., [11] and Osadalor and Okosun, [58] who all reported lower GSH levels in leprosy patients compared to controls. The reason for this is not clear.

Interestingly, there was significant negative correlation between GSH levels and duration of treatment. Decrease in GSH levels were observed with increase in treatment duration showing induction of oxidative stress with increased duration of MDT and only the MDT group had higher OSI and 8-OHdg compared to the controls. This may be due to the free radical producing effect of Dapsone, one of drugs in the MDT regimen [58]. Schlacher et al., [59] showed by their study that hydroxylation of Dapsone by hepatic cytochromes generate the metabolites (N-hydroxylamine DDS-NHOH and monoacetyl-hydroxylamine MADDS-NHOH) that produces free radicals by electron transfer. It may also have been utilized for replenishment of other crucial antioxidants such as vitamin E and C, which would get oxidized during the course of their antioxidant action [57]. They also suggested that MDT can thus reduce the activity of some antioxidant enzyme causing ROS accumulation and thereby exhausting the production of adaptive oxidants defenses, resulting in a higher oxidative stress index, lipid peroxidation as well as higher 8-OHdg levels in this group. The higher 8-OHdg suggests a predisposition of those undergoing treatment to oxidative DNA damage.

Our findings suggest that the increased oxidative stress observed may therefore be due to the body’s response to active infection; depletion of antioxidants defenses following therapy in the MDT and perhaps continued induction of free radicals by M. leprae antigens even after completion of therapy [46]. The increased OS observed in these patients can mediate inflammatory episodes, organ damage, depressed cell mediated immune response and degeneration of nerves in leprosy patients [12].

A limitation of this study was that we did not estimate the M. leprae bacterial load as this may have helped in shedding more light on the results obtained from those relieved from treatment i.e. to ascertain if there was persistence of infection. In addition, those relieved from treatment were significantly older than both the controls and those undergoing treatment. Though we tried to adjust for age, it may affect the interpretation of our result as age is a risk factor for cardiovascular disease and oxidative stress. Another limitation was the fact that we had no treatment–naïve group because this was not allowed by the hospital’s protocol because of the highly infectious nature of the disease. But it would have helped us observe the pattern of changes in the assessed analytes before, during and after treatment.

Conclusion

This study shows that there are lower levels of total antioxidant capacity and higher levels of total plasma peroxide, malondialdehyde in leprosy patients undergoing multidrug therapy and those relieved from treatment and higher levels of 8-OHdg and oxidative stress index in leprosy patients undergoing multidrug therapy. This is indicative of increased oxidative stress, in multibacillary leprosy patients irrespective of drug treatment status and increased oxidative DNA damage in those undergoing treatment. Leprosy patients relieved from treatment may be susceptible to cardiovascular events as shown by higher levels of VLDL-cholesterol, triglycerides and atherogenic index of plasma observed in that group compared to controls. Antioxidants supplementation may be beneficial in the treatment of leprosy to protect against the effects of oxidative stress and clinical follow up on patients relieved from treatment may also be necessary to monitor health status and prevent development of cardiovascular events.

(XLSX)

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23 Jun 2020

Dear Dr. Bassey,

Thank you very much for submitting your manuscript "Cardiovascular Risk Factors and Markers of Oxidative Stress and DNA Damage in Leprosy Patients in Southern Nigeria." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Alberto Novaes Ramos Jr, M.D., M.P.H., Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: To strengthen the work, it is necessary to align the analyzes carried out with a well-defined hypothesis and objective. I suggest that the authors include the purpose of the study in the introduction. In lines 102-105 the authors specify details about the collection of information and anthropometric data, however, they do not mention the measurement of blood pressure and the calculation of the waist-to-hip ratio. I suggest including this information as well as the reference values ​​associated with it. The absence of reference values ​​for the evaluated markers made it difficult to read and analyze the tables, so I suggest the authors to detail this information. Exclusion criteria topics can be added to the inclusion criteria topic. Lines 108-112 repeat some of the information mentioned in the previous topic (Study design and subject selection). I suggest that the authors review the need for repetition of information. In the statistical analysis, the authors do not inform whether they performed the assessment of the normality of the data and which test was used. This information must be clear since it supports the choice for parametric tests. I also suggest that the authors specify which regression analysis was used.

Reviewer #2: The objectives and hypothesis are clearly stated and the study design is appropriate to address the objective. Overall, the population is clearly described. No sample size calculation was given, but there seems to be a sufficient number of participants in each group (n = 30). The statistical analysis support the conclusions, however it is not clear whether the groups have similar characteristics (e.g. age, sex distribution). Ethical permission has been received and this was reported.

Reviewer #3: Well defined and nicely described methodology section of the study

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results follow the analysis plan. I recommend the authors briefly explore the differences between groups (MDT, RFT, and controls) for the results presented in Tables 1 and 2.

Table 1, which presents the sociodemographic data of the study subjects, did not include information on the age variable. The inclusion of this variable is relevant, as it is an important factor in the analysis of cardiovascular risk and oxidative stress.

The analysis of the waist-to-hip ratio without stratifying according to gender makes difficult to interpret since it has different reference values ​​between men and women.

Reviewer #2: The analysis match the analysis plan. The results are presented, but there is some information missing in table 1 (e.g. compliance) and the scale/value of some variables is not clear (e.g. duration of treatment in days/weeks/months?). The quality of the tables is sufficiently.

Reviewer #3: Nicely described results section as well as nicely presented analysis of results.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The discussion presented is interesting and states the limitations and generalizations of the study.

In lines 263-269, the authors discuss the role of pro-inflammatory cytokines in decreasing HDL levels. Since the patients included had multibacillary leprosy, the literature refers that the immune response elicited produces predominantly cytokines from the Th2 pattern. Studies have shown that TNF-α levels are increased in PB lesions and episodes of reverse reaction. These information should be taken into consideration when elaborating on the topic.

Reviewer #2: The conclusions are supported by the data presented and are clearly stated. The limitations of the analysis are clearly described and the authors show a great understanding of the topic. The public health relevance is addressed and recommendations are made for interventions.

Reviewer #3: Conclude the study section very good and given a key message for leprosy control program.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Abstract

Remove the abbreviation ROS, as the word is mentioned only once in the summary;

The abbreviations BMI, TC, WHR, and AIP have no mention of the original word.

Introduction

Line 45-46: specify the period to which the total number of people infected with leprosy refers to the sentence;

Line 52: present the terms referred to in the abbreviation ROS.

Methodology

Line 126: add the abbreviation FPG to the term fasting plasma glucose.

Results

Line 182: mean BPD values ​​were significantly lower in the MDT group when compared to the control group;

Line 185: correct the p-value for the TAC.

Reviewer #2: Minor revisions are needed for the methods, results and discussion.

I recommend to make the following revisions:

Methods:

Please describe what materials and methods you used. For example in line 118 'whole blood was collected' how did you do this and with what type of material from what company?

Also for methods that are described elsewhere like line 126 'method of Trinder' did you use the exact same material from the exact same company and distribution center?

What do you mean with minor modifications in line 141?

Please provid details on your protocols. For example in line 122 'frozen until assayed' at what temperature, -80 or -20?

Specify the ELISA kit you used and from what company, to do this correctly you need to give the companies full name, the city/state the company is located and the country the company is located and put this within brackets behind the material. So for example: Agisera's ELISA kit no:12345 (Agisera, Vännäs, Sweden).

This also applies to the software you use SPSS in line 155 and Microsoft in line 160

Results

Table 1: Did you test for differences between the groups, for example in gender distribution? And what about the age of the patients? It seems like there might be a difference in the gender distribution, did you check whether this affected your results and did you correct for this accordingly in your results?

Table 1: Not necessary to put disease form and HIV in it, since you selected for MB patients and for no other ilnesses, right?

Table 1: I miss the compliance in the table, this is described in line 166.

Table 1: how did you assess physical deformities, on what scale? This is also not described in the methods.

Table 2: Are the Calf F and Crit. F really needed?

Table 4: The duration of treatment is in days/weeks/moths/years?

Discussion:

Line 250 : 'both forms' what forms is meant by this. Be careful with the different 'forms' of leprosy, leprametous and MB are different terms since they belong to different classification systems but these terms overlap.

Line 365 - 368: What might be causing these different results?

Reviewer #3: Not required

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: It is a relevant study that contributes to the improvement of care for patients with leprosy by bringing evidence about the increase in cardiovascular risk and oxidative stress in MB patients in Nigeria. It adds knowledge about the theme and points to the need to monitor cardiovascular comorbidities in patients on MDT and released from therapy.

Reviewer #2: Overall, a very interesting manuscript! The introduction and discussion are well-written and show great understanding of the topic. This is very valuable since it is the first study in Africa evaluating oxidative status in leprosy patients, while leprosy is still prevalent in parts of this continent. The results are very interesting and relevant and the study has been performed according to ethical guidelines.

Reviewer #3: The manuscript Number PNTD-D-20-00597, entitled "Cardiovascular Risk Factors and Markers of Oxidative Stress and DNA Damage in Leprosy Patients in Southern Nigeria”. This is an interesting and exhaustive article on the cardiovascular risk factors in leprosy patients. The article is relatively well written. However, there are various fallacies in this article.

I would like the authors to consider the following points to make before this article can be processed further.

Comments on the manuscript PLOSNTD- major revision

01. How was the Multibacillary (MB) leprosy cases diagnosed?

02. The treatment for MB leprosy cases should be described in detail, including dosage and duration.

03. How was the cardiac evaluation done?

04. Was ECG and Echocardiography as well as X-ray chest done for evaluation of cardiac status?

05. What could have been the exact cause of oxidative stress and DNA damage?

06. Amyloidosis is an important cause of cardiac, renal and hepatic deterioration in MB leprosy patients. So mention has to be made of this entity in the discussion section.

07. In the introduction section, Epidemiology of the disease should be briefly described, including the number of cases worldwide as well as in Nigeria.

08. Mention has to be made about the leprosy elimination program in the world as well as in Africa and Nigeria in the discussion section.

09. The English and grammatical errors need to be corrected.

All the above mentioned points need to be taken care of before further processing.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

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Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

8 Jul 2020

Submitted filename: PLOS Neglected Tropical Diseases response to reviewers.docx

Click here for additional data file.

1 Aug 2020

Dear Dr. Bassey,

Thank you very much for submitting your manuscript "Cardiovascular disease Risk Factors and Markers of Oxidative Stress and DNA Damage in Leprosy Patients in Southern Nigeria." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Alberto Novaes Ramos Jr, M.D., M.P.H., Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Yes.

Reviewer #2: The methods are much more clear now that details have been added on the procedures and materials.

Reviewer #3: Described methodology of the study is well defined.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Yes.

Reviewer #2: As expected, the socio-demographic characteristics of the groups significantly differ. These characteristics can have major impact on the outcome parameters measured in this study. The increased aged in the group that is relieved from therapy can for example affect the results significantly. If I understand it correctly, these characteristics have not been taking along in the analysis of the outcome parameters, while they should be in my opinion. In addition, the paragraph describing table 2 and 3 (line 207-226) is really hard to read because of all the p-values in the text. Finally the last parameter in table 2 'duration relieved from therapy' is probably displaced, because now it is placed under the group that still receives therapy.

Reviewer #3: Results section is nicely presented.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes.

Reviewer #2: Only few limitations have been described, while this study has multiple. Especially the fact that the characteristics of the groups significantly differ and that this might affect the results is important to mention in the discussion. Please elaborate the limitations in the discussion.

Reviewer #3: The conclusion section of the study is excellent and forward a key message for leprosy control program.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: The manuscript needs formatting, in some sentences, there is an excess of periods (lines 201, 300), excessive use of spaces (lines 175,133, 136, etc.) and a different color in the text (269-273).

Reviewer #2: Please correct for the differences between the groups a priori (e.g. age, deformities) during your analysis of the outcome parameters. There is clearly a link between several cardiovasculair risk factors/markers of oxidative stress and age. So if the groups differ so significantly (p = 0.001) you can not 'just' compare the outcome parameters of these groups as you did in table 3. Please also discuss this in the discussion within the limitation section.

Reviewer #3: Not required

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors did a great job of reviewing and endeavored to improve the manuscript. The reviewers' suggestions were taken into account, and the doubts raised were clarified.

Reviewer #2: No comments

Reviewer #3: The manuscript Number PNTD-D-20-00597, entitled "Cardiovascular Risk Factors and Markers of Oxidative Stress and DNA Damage in Leprosy Patients in Southern Nigeria”. The revised article is relatively well written.

--------------------

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.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

2 Aug 2020

Submitted filename: PLOS Neglected Tropical Diseases response to reviewers 2.docx

Click here for additional data file.

26 Aug 2020

Dear Dr. Bassey,

We are pleased to inform you that your manuscript 'Cardiovascular disease Risk Factors and Markers of Oxidative Stress and DNA Damage in Leprosy Patients in Southern Nigeria.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Alberto Novaes Ramos Jr, M.D., M.P.H., Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Since the authors included a new analysis adjusted for age, it is necessary to give more details in the methodology section.

Reviewer #2: Methods are clear now

Reviewer #3: Method section of the study are well defined and nicely described.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The other reviewers pointed out that the age variable could be a possible confounding factor in the study. The authors then performed a new analysis adjusted for age. Therefore, there is now a need to include the results of this new analysis in a supplementary file or even throughout the text.

Reviewer #2: Results are correct now

Reviewer #3: Results section of the study is nicely presented.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes.

Reviewer #2: Limitations have been described properly now

Reviewer #3: Conclude of the study is section very good and given a key message for leprosy control program

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: line 452 - change subtitle formatting

line 348 - where it reads "It may also be important to note that the increase in may (...) "- a word seems to be missing from the sentence.

Reviewer #2: No

Reviewer #3: Not needed

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: No comments.

Reviewer #2: I recommend to accept this manuscript

Reviewer #3: The manuscript Number PNTD-D-20-00597, entitled "Cardiovascular Risk Factors and Markers of Oxidative Stress and DNA Damage in Leprosy Patients in Southern Nigeria”. This is an interesting article and relatively well presented.

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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.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

2 Oct 2020

Dear Dr. Bassey,

We are delighted to inform you that your manuscript, "Cardiovascular disease Risk Factors and Markers of Oxidative Stress and DNA Damage in Leprosy Patients in Southern Nigeria.," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases