A double-blind, randomized controlled trial to examine the effect of Moringa oleifera leaf powder supplementation on the immune status and anthropometric parameters of adult HIV patients on antiretroviral therapy in a resource-limited setting.

BACKGROUND: People living with HIV (PLHIV) in resource-limited settings are vulnerable to malnutrition. Nutritional interventions aimed at improving food insecurity and malnutrition, together with antiretroviral therapy (ART), could improve treatment outcomes. In Nigeria, there is a high awareness of the nutraceutical benefits of Moringa oleifera. Thus, this study aimed to evaluate the effects of Moringa oleifera leaf supplementation on the CD4 counts, viral load and anthropometric of HIV-positive adults on ART.
METHODS: This was a double-blind, randomized study. Two hundred HIV-positive patients were randomly allocated to either the Moringa Oleifera group (MOG) given Moringa oleifera leaf powder or the control group (COG) given a placebo. Changes in anthropometric parameters [weight; body mass index (BMI)] and CD4 cell counts were measured monthly for six months, while HIV-1 viral loads were measured at baseline and the end of the study for both groups.
RESULTS: Over the study period, the treatment by time interaction shows a significant difference in CD4 counts by treatment group (p<0.0001). A further estimate of fixed effects showed that the CD4 counts among MOG were 10.33 folds greater than COG over the study period. However, the viral load (p = 0.9558) and all the anthropometric parameters (weight; p = 0.5556 and BMI; p = 0.5145) between the two groups were not significantly different over time. All tests were conducted at 95CI.
CONCLUSION: This study revealed that Moringa oleifera leaf supplementation was associated with increased CD4 cell counts of PLHIV on ART in a resource-limited setting. Programs in low-resource settings, such as Nigeria, should consider nutritional supplementation as part of a comprehensive approach to ensure optimal treatment outcomes in PLHIV.

Introduction

The HIV and AIDS epidemic is a major pandemic that affects millions of people globally. The UNAIDS Global AIDS Update 2019 reported that 74.9 million people have become infected with HIV since the start of the epidemic, with 32.0 million deaths from AIDS-related illnesses [1]. Nigeria has the second-largest HIV epidemic worldwide [2]. In 2018, 130,000 new infections and 53,000 AIDS-related deaths were recorded. Nigeria alone accounts for more than half of the new infections and deaths from AIDS-related illnesses in the western and eastern Africa region in 2017 [3]. This high mortality is probably attributed to the large population size of Nigeria compared to other countries in the region [3].

Considerable progress has been made in providing global access to antiretroviral therapy (ART), with 23.3 million people accessing therapy worldwide [4]. ART has greatly reduced AIDS-related mortality and morbidity and increased the life expectancy of people living with HIV and AIDS (PLHIV) [5]; however, it has led to other consequences, including malnutrition [6]. PLHIV are vulnerable to malnutrition due to intestinal damage, which causes impaired nutrient absorption and reduced food intake from vomiting and painful swallowing [7]. Furthermore, malnutrition could result from food insecurity and the side effects of ART, such as appetite loss and abdominal pain [7]. The adverse effects of HIV and malnutrition on the immune system are similar in that they both reduce CD4 and CD8 T-lymphocyte numbers [8], which eventually increase susceptibility to opportunistic infections. Opportunistic infections and malnutrition can affect intake, absorption, and metabolism of food, worsen disease progression [7, 9] and increase HIV-related mortality [6].

PLHIV are encouraged to consume healthy diets rich in essential amino acids, unsaturated fats, and micronutrients at the recommended daily allowance (RDA) to achieve an adequate nutritional status vital for health and survival [10]. Unfortunately, several studies reported a poor diet intake with inadequate nutrients among PLHIV in Sub-Saharan Africa, including Nigeria [11, 12]. A study conducted in Nigeria reported significant malnutrition in early HIV infection before ART initiation [13].

Malnutrition is a public health challenge in Nigeria; available data showed that the country has the second-highest burden of stunted children worldwide [14]. Two million children and 7% of women of childbearing age were also reported to suffer from severe acute malnutrition [14].

Moringa oleifera Lam (syn. M. ptreygosperma Gaertn.) is a species of the monogeneric family Moringaceae [15, 16]. It has been documented to contain many nutrients and bioactive compounds in literature [17, 18]. The leaves are the part of the plant mostly used and with several nutrients often deficient in malnourished PLHIV. It is a rich source of both macro and micronutrients and natural antioxidants source [19]. Moringa oleifera leaf powder is a novel, cheap, culturally acceptable, efficacious, and regionally produced plant and can reduce the malnutrition burden in Sub-Saharan Africa [19]. Furthermore, in Nigeria, Moringa oleifera use is promoted based on the commendation of its nutraceutical benefits, and the Nigerian Federal Government Raw Materials Research and Development Council (RMRDC) has been actively encouraging farming and consumption of Moringa oleifera [20]. Monera et al. reported the in vitro CYP3A4 inhibitory activity of Moringa oleifera leaf extracts, suggesting the potential for interaction with antiretroviral drugs. However, the in vitro data alone is insufficient to conclude the clinical significance of concomitant administration of Moringa oleifera with ART in PLHIV [21]. Moreover, the interaction between tenofovir/lamivudine/efavirenz and Moringa oleifera leaf powder has not been reported. No adverse clinical effects have been reported in the literature despite its widespread use and concomitant use by PLHIV.

Therefore, this double-blind, randomized study aimed to evaluate the effects of six months of Moringa oleifera leaf supplementation on the CD4 counts, viral load and anthropometric parameters of HIV-positive adults who were on ART in Kano State, Nigeria.

Methods

Study location

The study was conducted at the S. S Wali Virology Center at the Aminu Kano Teaching Hospital, Kano State (AKTH), Nigeria. AKTH is a tertiary health institution and referral center that operates a daily HIV clinic (5 days a week). It also serves as a center for clinical evaluation, laboratory tests, HIV counseling and testing (HCT), treatment, and care supported by the Federal Government and the Institute of Human Virology, Nigeria (IHVN) in partnership with its global partners, including the Centers for Disease Control and Prevention (CDC) and the Global Fund to Fight AIDS, Tuberculosis, and Malaria. The center attends to all patients with HIV infection diagnosed within the hospital or referred from outside the health facility.

Type of study and participants

The study was a double-blind, randomized control trial conducted between December 2017 and November 2018. Registered HIV-infected individuals receiving treatment and care at the S.S Wali Virology Center were invited to participate. Inclusion criteria for the study were: being HIV sero-positive, ≥ 18 years old, CD4 counts ≤ 500 cells/mm3, ART for at least three months (tenofovir + lamivudine + efavirenz combination), informed consent, and compliance with the study protocol. Exclusion criteria for the study were: known allergy or intolerance to Moringa oleifera or placebo (cornstarch powder), pregnancy, CD4 counts > 500 cells/mm3, presence of active opportunistic infection, and intake of micronutrient or natural health product supplements within 30 days of screening. For ease of monitoring, patients who lived outside Kano State, where the study was conducted, were excluded.

Sample size

The sample size was calculated to ensure detection of medium effect size (Cohen’s d = 0.5) [22] or 0.5 standard deviation in mean weight or CD4 by randomized control trial (RCT) arm with 90% power (1-β [type 2 error probability]) and 95% confidence (or 5% α error probability [type 1]), assuming a balanced 1:1 study design. A sample size of 172 patients was calculated, rounded up to 200 to give room for attrition. The sample size was calculated using G*Power version 3.1.9.2 [23].

Randomization

Block randomization was used to balance the groups throughout the enrollment period. PASS 12.0 software was used to develop the randomization list using Wei’s Urn algorithm by an independent statistician who held the randomization code. A random allocation sequence was generated to allocate and assign each patient to either the Moringa Oleifera group (MOG) or the control group (COG) when participants fulfilled the inclusion criteria and consented, with 100 patients in each group. All the research team members, including the principal investigator (PI) and the study participants, were blinded to the allocation of patients to the study groups.

Preparation for study

Fresh Moringa oleifera leaves were obtained from Prime Global Agricultural Industries Limited, Kano State, Nigeria. Fresh leaves were identified and authenticated by a botanist at the Department of Biological Science, Bayero University Kano (BUK), Nigeria. Confirmation of the taxonomic identity of the plant was achieved by comparison with voucher specimens kept at the Herbarium of the Department of Biological Sciences, BUK. The leaves were processed by HOMIP Spices and Foods Limited, Kano State, Nigeria. The procedure involved washing and drying the fresh leaves in a clean environment on a net mesh away from direct sun for days until it was completely dried. The dried leaves were cleaned, and the small branches were removed. The dried leaves were ground using a grinder and sieved using a 0.500 mm standard sieve (No. 35 mesh size) [24, 25] to obtain a fine powder. Fine Moringa leaf powder was stored in airtight containers.

The placebo was obtained by coloring cornstarch powder with chlorophyll [26]. It was manufactured and processed at Dala Foods Nigeria Limited, Kano State, Nigeria. Both the Moringa oleifera and the placebo were similar in presentation and were identically packaged to be indistinguishable. The interventions were packaged into small (15 g) sachets each. Thirty (30) individual sachets were further packaged in a bigger green-colored plastic bag to be used as supplements taken together with meals for one month. It was sealed, labeled, and stored in a dry place away from heat and humidity. Patients could simply put a sachet in the pocket or bag while going out for their daily activities. The supplements were taken together with meals.

Intervention

The interventions were provided in 15 g Moringa leaf powder sachets. Thirty sachets were given to the participants to represent one month prescription, and they were directed to divide each sachet into three and use it thrice daily (5 g), adding it into meals [27, 28]. They were asked to maintain their regular diet and not consume Moringa oleifera in any form from other sources during the study period.

In Kano State, home visits of participants by research members to ensure adherence was not convenient for fear of stigmatization by family members. Thus, adherence was monitored by biweekly phone calls to the patients and interviewing them during their monthly visits to evaluate compliance.

Data collection

At the first visit, the research team interviewed the patients to obtain socio-demographic information, patient history, and other relevant information, including dietary information. A trained nurse at the virology clinic and a trained research assistant were responsible for all anthropometric measurements and data collection under the supervision of the PI. Weight was measured to the nearest 0.1 kg using a digital weighing scale (Tanita HD-372, Tanita Corporation, Tokyo, Japan), with participants wearing light clothing and without shoes. Height was measured to the nearest centimeter using a stadiometer (Seca 217, Seca Gmbh and co. KG., Hamburg, Germany). Body mass index (BMI) was calculated as the weight in kilograms divided by the square of height in meters. Anthropometric parameters were measured at baseline and each monthly visit.

All laboratory evaluations were performed by a trained phlebotomist at the President’s Emergency Plan for AIDS Relief (PEPFAR) laboratory of the S. S Wali Virology Center at AKTH. The CD4 count was tested using a Partec flow cytometer (Partec, Munster, Germany). Five (5 ml) venous blood samples were aseptically collected from each study participant. Briefly, equal volumes (20 μL) of CD4 PE antibody and ethylene diamine tetraacetic acid blood were mixed and incubated for 15 min, and 800 μL of CD4 buffer was added before reading in the cell counter [29]. The viral load was quantified by polymerase chain reaction (PCR). Ten (10 ml) of venous blood samples were aseptically collected from each study participant. The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2.0, manuals (Roche Diagnostics GmbH) was used as the standard operating procedure [30, 31]. The CD4 test was conducted at baseline and each subsequent monthly visit for each study participant, while the viral load test was conducted twice, at baseline and after the sixth month.

Study outcomes

The outcomes assessed were changes in immune status (CD4 cell count and viral load) and changes in anthropometric parameters (weight and body mass index [BMI]) and from baseline to the sixth month.

Data analysis

The data input was done in Microsoft excel and exported into SPSS and SAS statistical software for analysis. Findings from the analysis were reported in frequency tables, charts and descriptive analysis was done to estimate mean and standard deviation. Normality test for the data was conducted using Kolmogorov-Simonov and Shapiro-Wilk tests. Data which were not normally distributed were transformed through Box-Cox transformation. Independent t-test was used to determine the significance of mean difference in immunological and anthropometric parameters between the two groups at each stage of the experiment. To further confirm variability between the two groups, a repeated measure linear mixed effect model analysis was deployed to determine the difference in immunological and anthropometrics outcomes between the treatment groups overtime. An exploratory analysis was done to evaluate the influence of socio-demographic characteristics on the immunological and anthropometrics outcomes of the treatment groups over the study period. All statistical tests were carried out at 95% Confidence Interval.

Ethical considerations

This study was reviewed and approved by the ethics committee of Aminu Kano Teaching Hospital (AKTH) Kano, Nigeria (reference number NHREC/21/08/2008/AKTH/EC/2012), and the Biomedical Research Ethics Committee of the University of Kwazulu-Natal Durban, South Africa (reference number BFC294/16). The study was registered with the Pan African Clinical Trial Registry (identification number PACTR201811722056449). The study complied with the principles outlined in the Declaration of Helsinki [32]. All participants provided oral or written informed consent before enrolling them in the study. The procedures of the study, together with the aims, were explained to the participants. Participants were also informed of their right to withdraw from the study at any time.

Results

Participants flow

Fig 1 shows the flow chart of participants in the study. Four hundred and ten patients were screened and assessed for eligibility. Two hundred and ten patients were excluded (204 did not meet the inclusion criteria for the study, and 6 refused to participate). Two hundred patients were randomized into two groups. One hundred patients were randomly selected and allocated to the group receiving MOG, and 100 patients were randomly assigned to the group receiving COG. In the MOG, 8 patients were lost to follow-up, and 3 discontinued the intervention. In the COG, 9 patients were lost to follow-up, and 3 discontinued the intervention. Overall, 177 patients (89 and 88 in the MOG and COG, respectively) completed the 6-month study.

Fig 1

Flow chart of participants.

Nutritional contents of Moringa oleifera leaves powder

The nutritional content of a 100 g Moringa oleifera leaf powder (Nigerian ecotype) was analyzed using a South African National Accreditation System (SANAS) [33] accredited laboratory ASPIRATA Food and Beverage Laboratory [34]. Each 100 g contained an average of 28 g protein, 3.9 g total fat content (total saturated, monounsaturated, and polyunsaturated fatty acids), and 22 g carbohydrate. It contained 1791.82 mg calcium; 4879.26 mg potassium; 24 mg sodium; 2.88 mg Zinc and 37.78 mg iron (S1 & S2 Files).

Characteristics of participants at study inception

Table 1 shows the socio-demographic characteristics of the study participants at baseline. Participants in the MOG’s socio-demographic, socioeconomic, nutritional status, and immunological characteristics were similar to those in the COG at baseline. Females were predominant in both groups [MOG = 70 (78.7%); COG = 67 (76.1%)]. The majority were between 30 to 39 years of age in both groups [MOG = 37 (41.6%); COG = 36 (40.9%)]. The majority were married [MOG = 42 (47.2%); COG = 38 (43.2%)]. Islam was the predominant religion of participants in both groups [MOG = 64 (71.9%); COG = 66 (75%)] with more than half of participants belonging to the Hausa/Fulani ethnicity [MOG = 55 (61.8%); COG = 47 (53.4%)]. A few of the participants were without any form of education in either group [MOG = 15 (16.9%); COG = 16 (18.2%)]. The majority of the participants in both groups earned a monthly income below the minimum wage of ₦30,000 ($78.23) [MOG = 67 (75.3%); COG = 66 (75%)].

Table 1

Socio-demographic characteristics of participants.

Variables	MOG (%) (N = 89)	COG (%) (N = 88)	P-value
Gender
Males	19 (21.3)	21 (23.9)	0.689
Female	70 (78.7)	67 (76.1)
Age (years)
< 20	3 (3.4)	1 (1.1)	0.737
20–29	24 (27.0)	21 (23.9)
30–39	37 (41.6)	36 (40.9)
40–49	20 (22.5)	22 (25.0)
50–60	5 (5.6)	8 (9.1)
Marital Status
Married	42 (47.2)	38 (43.2)	0.838
Single	12 (13.5)	10 (11.4)
Divorced	19 (21.3)	20 (22.7)
Widowed	16 (18.0)	20 (22.7)
Religion
Islam	64 (71.9)	66 (75.0)	0.642
Christianity	25 (28.1)	22 (25.0)
Ethnicity
Hausa/Fulani	55 (61.8)	47 (53.4)	0.511
Yoruba	13 (14.6)	15 (17.0)
Igbo	9 (10.1)	15 (17.0)
Others	12 (13.5)	11 (12.5)
Educational Level
Primary	14 (15.7)	12 (13.6)	0.971
Secondary	27 (30.3)	24 (27.3)
Tertiary	20 (22.5)	21 (23.9)
Quranic	13 (14.6)	15 (17.0)
None	15 (16.9)	16 (18.2)
Occupation
Entrepreneur	15 (16.9)	10 (11.4)	0.840
Trader	23 (25.8)	25 (28.4)
Civil Servant	15 (16.9)	17 (19.3)
Artisan	19 (21.3)	17 (19.3)
Unemployed	17 (19.1)	19 (21.6)
Family Size
2–5	38 (42.7)	32 (36.4)	0.557
6–10	26 (29.2)	25 (28.4)
>10	25 (28.1)	31 (35.2)
Monthly Income(₦)
Not Indicated	11 (12.4)	6 (6.8)	0.672
< 30,000	67 (75.3)	66 (75.0)
30,001–60,000	6 (6.7)	10 (11.4)
60,001–90,000	1 (1.1)	1 (1.1)
90,001–120,000	3 (3.4)	2 (2.3)
>120,000	1 (1.1)	3 (3.4)

The baseline anthropometric and immunological characteristics of the study participants in both study groups showed a similar trend. The means of weight (kg) for both groups were [MOG = 63.8 (± 14.8); COG = 61.9 (±12.5)]. The mean BMIs for MOG and COG was 24.84 (± 4.76) and 23.75 (± 3.82), respectively. More than half of the patients had BMI within the normal range of 18.5–24.9 in both groups [MOG = (51.7%); COG = (58%)] while a significant number were overweight with BMI values of 25.0–29.9 [MOG = (30.3%); COG = (31.8%)] for both study groups (Table 2).

Table 2

Description of baseline anthropometric and immunological parameters between the two groups.

	Baseline
Parameters	MOG (n = 89)	COG (n = 88)	P-value
	Freq. (%)	Freq. (%)
Anthropometric
Weight (Kg)			0.361
Mean (±SD)	63.8 (±14.8)	61.9 (±12.5)
BMI (Kg/m2)			0.093
Underweight (<18.5)	5 (5.6)	5 (5.7)
Normal (18.5–24.9)	46 (51.7)	51 (58.0)
Overweight (25.0–29.9)	27 (30.3)	28 (31.8)
Obese (> 30.0)	11 (12.4)	4 (4.5)
Mean (±SD)	24.84 (±4.8)	23.75 (±3.8)
Immunological Parameters
CD4 Counts (Cells/μl)			0.547
< 350	46 (51.7)	38 (43.2)
≥ 350	43 (48.3)	50 (56.8)
Mean (±SD)	341.8 (±106.1)	352.34 (±126.0)
Viral load (RNA copies/ml)
<1000 (Undetected)	68	63	0.497
≥1000(Detected)	21	25

At baseline, the mean CD4 cell counts were statistically similar for both MOG and COG with values of 341.78 (± 106.06) and 352.34 (± 125.99) cells/μL, respectively (Table 2). The majority of the patients in both groups had an undetected viral load at baseline (MOG = 76.4% and COG = 71.6%) (Table 2).

A test of normality for the dependent variables was carried out using the Shapiro-Wilk and Kolmogorov-Smirnov tests. The data that were not normally distributed were transformed through Box-Cox transformation. The CD4 count was transformed through a lambda value of 0.5, weight by lambda value -0.1 and BMI by lambda value -0.2.

Effect of nutritional supplement intervention on immunological and anthropometric parameters

Table 3 shows the results of independent samples test for the difference in immunological parameters anthropometric between the MOG and COG. The mean CD4 count between the two groups was not significantly different throughout the period of measurement except at the 6th month. From baseline to the 6th month, there was no significant (P>0.05) difference in all the anthropometric parameters [weight; BMI] between the MOG and COG (Table 3).

Table 3

Bivariate analysis showing the differences in anthropometric and immunological parameters between the two study groups.

Parameters	Period	MOG (n = 89)	95% Confidence Interval		COG (n = 88)	95% Confidence Interval		F	P-value
		Mean (SD)	Lower	Upper	Mean (SD)	Lower	Upper
CD4 Counts	Baseline	341.78 (106.06)	319.43	364.12	352.34 (125.99)	325.64	379.03	4.88	0.55
	1st month	363.06 (127.91)	336.11	390.00	361.14 (130.28)	333.53	388.74	0.27	0.92
	2nd month	373.74 (130.79)	346.19	401.29	366.40 (144.47)	335.78	397.01	0.50	0.72
	3rd month	387.29 (134.61)	358.94	415.65	367.51 (142.24)	337.37	397.65	0.98	0.34
	4th month	401.51 (138.50)	372.33	430.68	368.78 (150.89)	336.81	400.76	0.24	0.14
	5th month	414.79 (144.02)	384.45	445.13	375.26 (152.18)	343.01	407.51	0.08	0.08
	6th month	425.75 (153.76)	393.36	458.14	373.44 (157.31)	340.11	406.77	0.02	0.03*
Weight	Baseline	63.83 (14.77)	60.64	66.73	61.94 (12.54)	59.45	64.82	1.62	0.36
	1st month	63.88 (14.89)	60.80	66.89	62.03 (12.92)	59.41	64.88	1.58	0.38
	2nd month	64.26 (14.76)	61.13	67.17	62.44 (13.26)	59.68	65.37	0.75	0.39
	3rd month	64.31 (14.93)	61.16	67.33	62.55 (13.36)	59.83	65.49	0.96	0.41
	4th month	64.47 (14.93)	61.29	67.44	62.73 (13.37)	60.04	65.65	0.92	0.41
	5th month	64.73 (15.00)	61.59	67.76	62.99 (13.38)	60.27	65.91	1.18	0.42
	6th month	64.71 (15.07)	61.54	67.82	63.16 (13.49)	60.48	66.19	1.09	0.47
BMI	Baseline	24.84 (4.76)	23.84	25.85	23.75 (3.82)	22.94	24.56	3.52	0.09
	1st month	24.86 (4.84)	23.84	25.88	23.78 (3.93)	22.94	24.61	3.25	0.10
	2nd month	24.99 (4.82)	23.98	26.01	23.92 (4.02)	23.08	24.78	2.06	0.11
	3rd month	24.99 (4.88)	23.97	26.02	23.96 (4.04)	23.11	24.82	1.76	0.13
	4th month	25.06 (4.87)	24.03	26.08	24.04 (4.10)	23.17	24.91	1.45	0.14
	5th month	25.16 (4.93)	24.12	26.20	24.14 (4.08)	23.27	25.00	1.78	0.14
	6th month	25.16 (4.93)	24.12	26.20	24.19 (4.09)	23.33	25.06	2.40	0.16

* Statistically significant difference between two groups.

In addition to the bivariate analysis test above, a linear mixed-effect model was used to examine the differences in anthropometric and immunological parameters between the MOG and COG. Table 4 shows the linear mixed effect model results showing the differences in the CD4 counts, viral load, weight and BMI between the two groups over the study period. An unstructured correlation matrix was assumed for the model analysis. For CD4 counts, the treatment by time interaction shows a significant difference in CD4 counts by treatment group over time (p<0.0001). A further estimate of fixed effects showed that the CD4 counts among MOG were 10.33 folds greater than COG over the study period. On the other hand, viral load (p = 0.9558) and the anthropometric parameters (BMI; p = 0.5145 and weight; p = 0.5556) between the two groups were not significantly different over time (Table 4).

Table 4

Linear mixed effects model framework showing the differences in immunological and anthropometric parameters between the treatment groups overtime.

	Estimates of Fixed Effectsa
	Parameter	Estimate	Std. Error	t	Sig.	95% Confidence Interval
						Lower Bound	Upper Bound
	Intercept	356.35	13.10	27.20	0.0001	354.29	376.09
CD Counts	MOG	10.33	2.65	3.89	0.0001*	5.12	15.54
	COG	0b	0
	Intercept	-1.07	0.31	-3.47	0.0007	-2.02	-0.99
Viral load	MOG	-0.005	0.09	-0.06	0.9558	-0.19	0.18
	COG	0b	0	.	.	.	.
	Intercept	61.92	1.47	42.02	0.0001	61.43	63.65
Weight	MOG	-0.05	0.08	-0.59	0.5556	-0.20	-0.11
	COG	0b	0	.	.	.	.
	Intercept	23.73	0.47	50.98	0.0001	23.61	24.32
BMI	MOG	-0.02	0.03	-0.65	0.5145	-0.08	-0.04
	COG	0b	0	.	.	.	.

* = statistically significant.

Fig 2 shows the chart depicting CD4 cell count mean measurements by treatment group over the study period. Over the six months study period, there was significant increase in mean CD4 cell counts for MOG while the mean CD4 cell counts for the COG was relatively constant.

Fig 2

Chart depicting CD4 cell count mean measurements by treatment group over the study period.

An exploratory analysis of the influence of socio-demographic characteristics on the changes in immunological and anthropometric parameters by treatment groups over time was computed. The analysis of the impact of socio-demographic characteristics on the changes in CD4 counts by treatment group over time showed that ethnicity (p = 0.0491) and family size (p = 0.0483) had a significant influence. However, the changes in CD4 counts by treatment group remained significantly different over time (p = 0.0001). None of the socio-demographic characteristics explored significantly influenced the viral load, BMI, and weight overtime. The changes in these parameters between the treatment groups over time were not significant after controlling for socio-demographic characteristics.

Discussion

This study examined the effect of the sixth month’s consumption of Moringa oleifera leaf powder supplement on the immunological profile (CD4 cell count and viral load) and anthropometric parameters (weight and BMI) of PLHIV that are on ART in Kano State, Nigeria. We studied 200 patients randomly divided into MOG (100 patients) and COG (100 patients). Our sample size was larger than that reported by Tshingani et al. (60 patients) in the Democratic Republic of Congo and Ogbuagu et al. (40 patients) conducted in Anambra State, Southeast Nigeria [10, 35].

Over the study period, a significant increase was observed in CD4 cell counts of the MOG participants than the COG using the linear mixed effect model. There was no significant difference in viral load between the two study groups. This improvement observed in the CD4 counts in the MOG was influenced by some socio-demographic characteristics of the study participants that include ethnicity and family size. However, the improvement was still detected regardless of their influence. This result suggests that Moringa oleifera leaf powder supplementation and ART effectively improved the CD4 cell counts of the study participants.

Conversely, the Moringa oleifera leaf powder supplementation intervention was not effective in improving the weight and BMI of the patients when compared to the COG over the study period. Furthermore, none of the socio-demographic characteristics explored was observed to significantly influence any of the anthropometric parameters overtime.

Moringa oleifera nutritional constituents and ART effect could be responsible for the increased CD4 cell counts in the MOG. Moringa oleifera leaves (Nigerian ecotype) analysis shows that they are rich sources of vitamins and micro-and macronutrients. Additionally, it contains minerals and trace elements reported to have multiple curative properties, improve the immune system, and act as strong antioxidants [17-19]. Furthermore, the dried leaves of Moringa oleifera have been documented as a good source of polyphenol compounds, such as flavonoids. Flavonoid consumption has been reported to offer body protection against chronic diseases associated with oxidative stress [18].

In addition, ART has proven effective in reducing morbidity and mortality related to HIV infection by reducing HIV viral load. This improved CD4 level is associated with a reduced number of opportunistic infections [36]. Our results agree with a similar study from Eastern Nigeria by Ogbuagu et al.. A significant increase in CD4 counts was observed in PLHIV receiving ART and supplemented their local meals with Moringa oleifera leaf powder for two months. Local meals were prepared using palm oil. The limited information offered by the study article prevented an in-depth analysis of the results. However, the study demonstrated the potential of Moringa oleifera of improving the CD4 count of PLHIV within short period of supplementing it with regular meals prepared using local food items [35]. Palm oil has been documented to contain palmitic acid which is a saturated fatty acid with health benefits [37].

The lack of significant change in BMI observed in MOG in our study could be due to the fact that few participants (n = 5) were underweight with BMI<18.5 kg/m2 at study inception. This class of people would probably have benefitted more in terms of improvement in BMI from the Moringa oleifera leaves supplementation due to the vast amounts of nutrients constituted.

Contrary to our results, Tshingani et al. did not report a significant difference in CD4 lymphocyte counts after six months of Moringa oleifera leaf powder supplementation between their study groups. This could be due to the small sample size. In addition, the presentation of their intervention in bags of 100 g could have reduced adherence, which resulted in a lack of significant increase in CD4 cell counts [10].

This study’s outcomes can be attributed to factors related to the study design. This includes being a double-blinded randomized trial, a larger sample size, and the presentation of the intervention in individual sachets representing daily dose. In addition, the involvement of the virology clinic ‘support group’ members improved patient monitoring and adherence to the study protocol. Nevertheless, future studies using a more diverse population of PLHIV are recommended.

The Moringa oleifera leaf intervention was not effective in decreasing the viral load of HIV-infected individuals accessing ART at the S.S Wali Virology Center. This could be attributed to some of the challenges encountered. The study protocol did not follow the standard operating procedure (SOP) of viral load monitoring conducted at the S. S Wali Virology Center. Viral load was monitored yearly for patients without any medical problems, whereas the study protocol was designed to have a viral load test conducted twice, at baseline and after six months of receiving the intervention. The study plan overcame this challenge. The use of viral load alone without CD4 cell counts to monitor treatment outcomes remains a challenge in resource-limited settings.

Further challenges encountered during the study also include participants’ reluctance to keep monthly appointments to the clinic for the study. This is because at the S. S. Wali virology center, ART drugs were dispensed to last for two to three months for patients with stable medical conditions. This challenge was alleviated with the bi-weekly telephone calls performed to monitor the study participants and remind them of their hospital appointments. The stipend given for transport fare after each monthly hospital visit assisted the study participants in keeping their appointments. This is because of the high poverty level in resource-limited settings such as Kano State and the poor socioeconomic status of the study participants. No other incentives or gifts were provided to participants.

Some limitations of this study must be noted. The distinguishable taste of Moringa oleifera could be a source of bias. Use of Moringa oleifera in capsules could forestall this limitation in future studies. In addition, the inclusion of patients who were only on one ART regimen (tenofovir + lamivudine + efavirenz drug regimen) limits the generalizability of our study findings. Lastly, the short duration of the study and compliance, which was monitored by the self-reporting of the study participants, are further limitations of the study.

Conclusion

This study revealed an association between Moringa oleifera leaf nutritional supplementation consumption and increased CD4 cell counts among PLHIV on ART in a limited resource setting. Programs in low-resource settings, such as Nigeria, should consider nutritional supplementation as part of a comprehensive approach to ensure optimal treatment outcomes in PLHIV.

CONSORT checklist.

(DOC)

Click here for additional data file.

Certificate of analysis Moringa oleifera powder.

(PDF)

Click here for additional data file.

Certificate of analysis Moringa oleifera powder_Minerals.

(PDF)

Click here for additional data file.

Detailed statistical analysis of data.

(DOCX)

Click here for additional data file.

Study protocol.

(DOCX)

Click here for additional data file.

18 Jan 2021

PONE-D-20-30776

A double-blind randomized control trial to examine the effect of Moringa Oleifera leaf powder supplementation on the anthropometric and immune status of adult HIV patients on antiretroviral therapy in a resource- limited setting

PLOS ONE

Dear Dr. Gambo,

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.

The manuscript has been evaluated by three reviewers, and their comments are available below. You will see the reviewers have commented on the importance of your work. However, the reviewers have also raised critical concerns and the manuscript will need significant revision before it can be considered for publication – you should anticipate that the reviewers will be re-invited to assess the revised manuscript, so please ensure that your revision is thorough. I have outlined some of the key concerns noted by the reviewers below, but you should respond all concerns mentioned by the reviewers in your response-to-reviewers document.

The key concerns noted by the reviewers relate to the study intervention and analysis. Specifically, the reviewers have suggested alternate approaches to the statistical analysis and presentation of results. Additionally, Reviewer 2 noted that the distinguishing taste of MOG may be considered a source of potential bias in this study. Finally, Reviewer 3 noted that inclusion was limited to participants on one ARV regimen, which limits the generalizability of the findings. These issues have limitations for the interpretation of the results and should be explored.

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Staff Editor

PLOS ONE

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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: No

Reviewer #3: Partly

**********

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

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: No

**********

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: *** General comments: ***

The manuscript presents interesting data from what appears to have been a well-run study.

However, the statistical analysis is quite idiosyncratic and fails to control for multiple comparisons. It should be redone.

In addition, the manuscript suffers from a variety of English syntactic and usage errors. These should be corrected. There are relatively few in the results section, they are more frequent in the discussion section, and they are numerous in the remaining sections. There are too many to list.

*** Specific comments: ***

1) The protocol is quite weak in its description of the statistical methods to be used for the data, as well as the hypotheses of interest and the specific tests or contrasts that were to be employed. This weakness appears to have carried over to the actual statistical analysis. The study design is fairly straightforward, so it is unclear why a better framework for evaluation could not be specified.

Having said that, the most important test specified in the protocol is the t-test of the two treatments at the last time point, so this should be presented in any case.

2) The current layout of the statistical analysis is quite idiosyncratic and simplistic. The following approach or equivalent should be used instead:

Test baseline differences via the t-test as already performed. This is analogous to a two-factor analysis of variance (ANOVA) with one fixed effect --- that is, treatment.

Assess the differences between groups in changes over time by using a linear mixed effect model framework. The fixed effects are treatment, time, and the treatment by time interaction. Include a correlation structure to account for the dependence structure caused by observing the same patient over time. Do not use a compound symmetry structure (equivalent to "classical" repeated measures) --- this is an unrealistic assumption here. Instead, either use an autoregressive correlation structure such as AR(1) (since time points are equally spaced) or use a completely unstructured correlation matrix.

The test of the interaction of treatment by time provides the answer to the question of whether the response profile over time differs between treatments. If there is no interaction effect, that is not necessarily the end of the road, but it is arguably of primary interest.

Next, perform post hoc comparisons using an adjustment for multiple comparisons such as the Tukey-Kramer adjustment. It is up to the authors, they can perform an analysis of either simple effects (comparison between treatments at each time point or comparison among time points within each treatment) or an analysis of all pairwise comparisons. Note that the test of the interaction effect is not technically required to be statistically significant for these tests to be performed, though this is the usual procedure.

All linear models should be assessed for fit using graphical analysis of the residuals to check for symmetry, homoscedasticity, and independence. The Shapiro-Wilk or Kolmogorov-Smirnov test can be used, but is probably of less importance.

If transformations are required, please report all transformations attempted. Even if fit is marginal on the original scale, if the results are qualitatively similar to a fit on a transformed scale then it may be best to report analysis on the original scale for interpretation.

For completeness's sake, since the protocol specifies it, also include the t-test of the last endpoints. However, this is probably less powerful than the result that will be obtained from the linear mixed effects model approach.

3) The evaluation of socio-demographic characteristics (Line 270 forward) should be redone. This should be done in the framework described above, as an exploratory analysis. These covariates can be entered as main effects in linear mixed effects model described above. This will yield answers to the question of whether the treatment effect remains if the effect of the covariate is statistically corrected, which is probably the question that the authors have in mind.

4) In some cases, it is difficult to understand what exactly is being presented. For example, what does Table 5 present? What are the "differences" here? Each table and figure should have a caption that clearly describes its contents.

5) Figure 2 is really terrible and should be replaced with a standard figure that shows the behavior of the expected marginal means over time for each group.

6) Lines 317-318: This conclusion seems far too strong for this paper. Has it really been proved to this degree?

7) Lines 338-344: The authors do not disclose whether similar claims were made by COG patients. This paragraph seems really close to misinformation as it is currently worded. If patients from both treatments reported these claims, then how can they be attributed to Moringa oleifera? All such claims in the discussion need to be backed up by data analysis in the results. Where is the presentation of these results?

8) The methods section lacks critical details. As an example, on Line 175 the authors state that viral loads were measured "by the Laboratory scientist using Real-time PCR machine" (sic). Which machine? What was the protocol? Which laboratory? The same issue pertains to the CD4 counts.

9) Lines 403-405 overstates the results.

10) The conclusion and recommendation in Lines 417-121 do not seem to be fully supported by the manuscript.

Reviewer #2: 1. Moringa leaf powder packaged in sachets as used in the MOG potentially had a taste distinguishable from COG. This most likely violated the blinding process especially in a community where there was a “high awareness” level of Moringa’s usefulness as a nutraceutical. This should be considered as significant source of bias enough to dispute the conclusion of a causal relationship between the MOG intervention and the observed increase in CD4 count. A more firm conclusion could have been justified if the powder had been packaged in gelatin capsules which could mask the taste and also ensure more accurate and consistent dosing of the product.

2. While literature references were made, the claims that Moringa leaf samples contained essential and non-essential amino acids, wide range of vitamins, minerals, carotenoids, polyphenols, phenolic acids, flavonoids, alkaloids, glucosinolates, isothiocyanates, tannins and saponins were unsubstantiated in this particular study. Perhaps a description of whether the manufacturer Prime Global Agricultural Industries Limited routinely analyzed the samples for the claimed ingredients would have been useful.

3. Addition of the discussion of the potential of interactions between Moringa and antiretroviral therapy, particularly with efavirenz via alteration of liver metabolism would enrich the manuscript.

4. It is more scientifically acceptable to use the terms such as “study participants” or “HIV infected individuals” instead of “HIV patients”.

5. Sentences on line 174 and line 176 need reconstruction

6. There may still be more work before a causal relationship can be claimed between the consumption of Moringa leaf powder and observation of increased CD4 count. Review recommends that the conclusion should limited to an association between the intervention and increased CD4 count, siting the observed shortcomings of the study design.

Reviewer #3: The manuscript reports findings from a randomised controlled double blinded trial assessing the effect of Moringa oleifera (MO) leaf powder supplementation on weight, BMI, MUAC, CD4 count and viral load. This is an important study given that MO is widely consumed and is an important herb among people living with HIV. Based on the data, the authors conclude that only the CD4 cell counts improve significantly (p=0.03) after 6 months concurrent MO leaf supplementation and ART. The manuscript however has some technical issues in some aspects of the trial design and statistical analysis.

p11 Selection criteria

L105 - Inclusion of patients on only one regimen limits generalisability of the effect on CD4 counts to patients on other regimen. Why was this necessary?

L113 - Typically, randomisation follows screening and indicates a participant is enrolled into the study. However, under exclusion criteria authors state that patients taking micronutrient or natural health product supplements within 30 days of randomisation were excluded. Please clarify.

p12 Sample size

L122 - In the abstract the authors talk about significance of the mean increase of CD4 count from baseline at the 99% CI which is inconsistent with sample size justification based on a 5% margin of error, i.e. 95% CI.

p12 - 13 Intervention

L135 - How were the MO leaves positively identified? Was a botanist consulted?

L156 - What seive size was used to prepare the intervention? 5g weight seems too heavy for 1 teaspoon of MO leaf powder.

L158 - Specify that MO from other sources is what was disallowed.

p14

L174, L177 - specify the type and volume of blood samples collected and a brief explanation or reference to the details of the laboratory tests.

p15

L202 - It is the participants who provide consent to participant in the study, not the researchers. This presents a significant ethics issue for this study.

p18-19

Why was the change in anthropometric and immmunological parameters within group important to analyse statistically? Given the objectives of the study, it is the difference between the intervention and control groups that is relevant. The results section is crowded with results that may not be important. In addition, the change from baseline at the end of each month may be more useful than the change between each month. Consider revising the results section and highlighting only the difference in change from baseline between the intervention and control group. One figure and one table, with legends, and focusing on the key results will further enhance the section.

p24

L334 - There is no data on which the discussion on 'good adherence' is based. The strategy the authors describe on p13 L161 was to monitor adherence, rather than enhance or optimise it. No results are presented on the level of adherence. In fact a large portion of the discussion refers to perceptions from participants and other operational and implementation aspects that are core to the study and not presented in the results and are appearing for the first time as discussion.

p21

L407 - From the power calculation, the study was adequately powered, perhaps a more diverse population of PLWH rather than a larger sample size could be recommended? The recommendations should be based on the effect on MO on CD4 and not extend to issues of food security and nutrition which have not been demonstrated by the current study.

Several challenges and limitations are cited. To what extend were participants reluctant to keep clinic appointments? What was the effect on study outcomes and how was that accounted for in the analysis?

The write up also has some grammatical errors and could benefit from revision by an English editor. Some comments that may be helpful

Page 1

L1 - 'oleifera' is always written in lower case and itilised. Correct throughout the manuscript.

L2 - Be consistent with the term used for study population. Ideally, use people first language such as 'people living with HIV (PLWH)' rather than HIV patients or HIV infected patients.

Page 9

L46 - Change accountable to accounts

L48 - revise grammar

L71 - 'leaf' powder

L72 - Sub-Saharan

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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.

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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: No

Reviewer #2: No

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.]

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4 May 2021

All comments have been responded to and attached as 'Answers to reviewers comments'.

Submitted filename: Answers to Reviewers comments_ PLOS ONE.docx

Click here for additional data file.

24 May 2021

PONE-D-20-30776R1

A double-blind randomized control trial to examine the effect of Moringa Oleifera leaf powder supplementation on the anthropometric and immune status of adult HIV patients on antiretroviral therapy in a resource- limited setting

PLOS ONE

Dear Dr. Gambo,

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 after your first revision. Therefore, we invite you to submit a further revised version of the manuscript that addresses the outstanding statistical issues raised during the current review process.

Please submit your revised manuscript by Jul 08 2021 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:

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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. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tsitsi Grace Monera-Penduka, PhD, MSc, MPhil, BPharm Hons

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

The authors have addressed the grammatical and other issue raise satisfactorily save the statistical issues. They should revisit additional comments from the current review and address them.

[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)

**********

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

**********

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

Reviewer #1: I Don't Know

**********

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

**********

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

**********

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: The authors appear to have at least partially implemented some changes to the statistical analysis. However, it is not possible to tell whether the analysis was performed correctly due to the lack of detail in the description of the statistical analysis and in the results shown. From what can be seen, it appears that the analysis was not specified correctly; or, if it was specified correctly, it was not reported correctly.

Also, the authors should put some energy into evaluating whether the different types of analysis performed produce similar results. My suggestion to keep the planned analysis was because as a rule all pre-planned analyses should be presented unless completely inappropriate. However, that means that there will be potentially conflicting results, perhaps due to the effects of statistical correction, perhaps due to increased or decreased power, or perhaps due to different assumptions.

In fact, this might result in relegating some part of the current work into a supplementary appendix on the grounds that it is less germane.

The covariate analysis that is presented is also difficult to assess. The model structure (as in the other case) is not clearly specified. If transformations were used, they are not described. Note that "transformation" does not mean changing data format from wide to long format. Instead, it means such things as logarithmic transformation or other functional transformations, aggregation, or recoding. Simple manipulation of data shape is irrelevant to the manuscript because it is only a technical requirement of the data analysis software.

As initially mentioned in the first review, all linear model analysis must be subjected to goodness of fit evaluation before interpretation. This is described in brief in the first review and does not appear to have been performed.

My current recommendation is to obtain the services of a consulting statistician to work through the data analysis and review comments and to put everything onto a solid and consistent footing.

**********

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: 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.

3 Oct 2021

All comments have been addressed

15 Dec 2021

A double-blind, randomized controlled trial to examine the effect of Moringa oleifera leaf powder supplementation on the immune status and anthropometric parameters of adult HIV patients on antiretroviral therapy in a resource-limited setting.

PONE-D-20-30776R2

Dear Dr. Gambo,

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.

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17 Dec 2021

PONE-D-20-30776R2

A double-blind, randomized controlled trial to examine the effect of Moringa oleifera leaf powder supplementation on the immune status and anthropometric parameters of adult HIV patients on antiretroviral therapy in a resource-limited setting

Dear Dr. Gambo:

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PLOS ONE