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. Author manuscript; available in PMC: 2024 Apr 1.
Published in final edited form as: Acta Trop. 2023 Feb 10;240:106860. doi: 10.1016/j.actatropica.2023.106860

Red palm olein-enriched biscuit supplementation lowers Ascaris lumbricoides reinfection at 6-month after anthelmintic treatment among schoolchildren with vitamin A deficiency (VAD)

Pei Yee Tan 1,2, Radhika Loganathan 1,*, Kim-Tiu Teng 1,*, Soo Ching Lee 3, Johari Syahirah Nadiah Mohd 2, Kanga Rani Selvaduray 1, Romano Ngui 2, Yvonne Ai-Lian Lim 2,*
PMCID: PMC10241531  NIHMSID: NIHMS1875720  PMID: 36775004

Abstract

Notwithstanding the global efforts made to control intestinal parasitic infections, soil-transmitted helminth (STH) infections are still one of the most prevalent infections globally, especially in developing countries. A double-blinded, randomised controlled trial was conducted on 343 primary schoolchildren (8–12 years old) with vitamin A deficiency (VAD) in rural areas of Malaysia to investigate the effects of red palm olein (RPO)-enriched biscuits on STH reinfection rates and infection intensity. The effects of the RPO-enriched biscuits (experimental group, n=153) and palm olein (PO) biscuits (control group, n=190), were assessed at 3- and 6-month after the administration of complete triple-dose albendazole (one dose of 400 mg for three consecutive days). The overall STH infection rate at baseline was recorded at 65.6%. At 6-month, a significantly lower reinfection rate of A. lumbricoides was observed in the experimental group (35.3%) compared to the control group (60.0%) (P<0.05), and a significant reduction in faecal egg count (epg) of A. lumbricoides was observed in the experimental group from baseline (P<0.001), but no significant reduction was observed in the control group. No significant differences in the reduction of infection intensities of T. trichiura and hookworm were observed between experimental and control groups at 3- and 6-month (P>0.05). These findings suggest the potential beneficial effects of RPO-enriched biscuit supplementation on the reinfection of A. lumbricoides, which could be attributed to its high carotenoids content by enhancing host immune response and mucosal epithelium integrity. However, further studies are warranted to confirm whether RPO supplementation could result in similar parasite-specific beneficial effects in other community settings, as well as to explore the underlying mechanisms.

Keywords: Red palm olein, soil-transmitted helminth, reinfection, vitamin A deficiency, randomised controlled trial

1. Introduction

Soil-transmitted helminth (STH) infections are one of the most widespread infections worldwide, affecting more than 1.5 billion individuals, accounting for 24% of the world’s population (World Health Organization., 2022). The main STH species that infect humans include whipworm (Trichuris trichiura), roundworm (Ascaris lumbricoides), hookworm (Necator americanus and Ancylostoma duodenale). School-aged children are one of the most vulnerable groups affected by STH infections, and it is estimated that over 571 million school-aged children in 102 countries were at risk of the infections and required preventive chemotherapy in 2015 (World Health Organization., 2016). STH infections often coexist with malnutrition and anaemia, and may negatively impact the physical growth, cognitive function, and academic performance of the children (Djuardi et al., 2021; Pabalan et al., 2018).

Mass deworming is regarded as a safe, effective, and economical approach to reduce worm burden (Lo et al., 2016). However, STH reinfection is common in endemic communities owing to the persistent exposure to the helminths, and the prevalence and infection intensities could return to the pre-treatment levels after six months duration (Al-Mekhlafi et al., 2008; Dunn et al., 2019). It has been shown that malnutrition can increase an individual’s susceptibility to parasitic diseases (Papier et al., 2014). Hence, a dual intervention strategy comprising nutritional supplementation and anthelminthic treatment has been suggested to address both undernutrition and STH infections (Yap et al., 2014). However, its effects on the reinfection of STHs are scarcely reported.

World Health Organization (WHO) recommends the co-administration of anthelmintic treatment and vitamin A supplementation as there are substantial geographic overlaps between STH infections and vitamin A deficiency (VAD) (Strunz et al., 2016; World Health Organization., 2004). Vitamin A supplementation in conjunction with deworming treatment was found to reduce A. lumbricoides reinfection in children, but the effect was less evident in stunted children (Payne et al., 2007). In contrast, a study among aboriginal schoolchildren living in communities with high endemicity of STH in Malaysia reported that vitamin A supplementation did not have any protective effect against STH reinfection (Al-Mekhlafi et al., 2014). Hence, due to the limited and inconclusive evidence available, more studies need to be conducted to explore the effects of nutritional supplementation on STH reinfection.

Red palm olein (RPO) is a rich source of health-promoting phytonutrients, including provitamin A carotenoids, which comprise mainly α- and β-carotenes, as well as tocotrienols, tocopherols, phytosterols, squalene, and coenzyme Q10 (Loganathan et al., 2017). Carotenoids are postulated to play an important role in immune function by cell-mediated and humoral immune responses (Chew and Park, 2004). Vitamin A is also known to play an essential role in enhancing immune function, and thus the immune-modulating effects of provitamin A carotenoids that are found abundantly in RPO could be demonstrated via their metabolism or conversion into retinol (vitamin A) in our body (Huang et al., 2018; Rühl, 2007). In view of the potential immune-enhancing effects of RPO, we aim to investigate the effects of the RPO-enriched biscuit supplementation on STH reinfection prevalence and infection intensity after anthelmintic treatment among primary schoolchildren with VAD in rural areas of Malaysia.

2. Materials and methods

2.1. Study area and participants

This study is part of a nationwide clinical trial that was carried out between April 2017 to June 2019 in ten national primary schools located in rural areas of five different states in Malaysia. The criteria for the selection of locations and schools were described previously (Tan et al., 2022). The main objective is to investigate the effects of RPO-enriched biscuit supplementation on vitamin A status among VAD children. Therefore, the sample size was determined based on an estimation of standard deviation at 0.3 μmol/L and true difference at 0.15 μmol/L for retinol concentration one year after the red palm oil supplementation conducted among schoolchildren in Burkina Faso (Zeba et al., 2006). A sample size of at least 280 schoolchildren (140 schoolchildren per arm) with a power of 95% and a two-sided significance level of 0.05 is required for the conduct of the nationwide intervention study based on an estimated drop-out rate of 40% in rural schools and a VAD prevalence of 4.5% in Malaysia as reported by Poh et al. (2013).

During the screening visit, 1164 schoolchildren were assessed for eligibility based on the following criteria: (1) 7 to 11 years old at the screening phase, (2) diagnosed with confirmed VAD or marginal VAD, which was determined by retinol concentration <0.70 μmol/L and 0.70 to <1.05 μmol/L, respectively (World Health Organization, 2012), and (3) not physically handicapped. In addition, the schoolchildren were excluded if they: (1) have oedema including severe acute malnutrition or gastrointestinal disorders, (2) are allergic to wheat- and/or gluten-containing foods and (3) are studying at standard six or 12 years old (because they would have completed primary school education during the commencement of intervention phase).

2.2. Study design and interventions

This study was a double-blinded randomised controlled trial, whereby the study sites were clustered by schools: in each state, experimental schools were given RPO-enriched biscuits while control schools were given PO biscuits. Randomisation was done based on permutation using a computer-generated randomisation list, with the students from different schools matched for VAD status, age, sex, and overall STH infection status upon randomisation.

After the screening for eligibility and STH infections examination at baseline, all the schoolchildren were administrated with three doses of 10 mL Vemizol suspension which contains 400 mg of albendazole (one dose per day for three consecutive days) in front of trained fieldworkers or teachers before the initiation of intervention, except those from Sarawak were given only one dose as they were found to have zero STH infection during the screening visit (unpublished data).

The schoolchildren were supplemented with biscuits prepared with RPO shortening (experimental group) or PO shortening (control group) with similar fatty acid composition (44% 16:0, 4% 18:0, 39% 18:1, and 10% 18:2), and they were required to consume all the biscuits given four days a week on schooling days in front of teachers, and in front of the guardians or field workers assigned during school holidays. A total of 4 types of biscuit flavours were formulated for a duration of 6 months and packaged in individual foil wrappers with identical packaging across the groups. A cookie manufacturing company was appointed to produce the biscuits with close monitoring by the research team throughout the production for quality control. On average, RPO-enriched biscuits contain higher total carotene but similar vitamin E contents (389 μg/ml carotenes and 523 μg/ml vitamin E per 36 g serving) when compared with PO biscuits (13 μg/ml carotenes and 504 μg/ml vitamin E per 36 g serving). Regarding the macronutrient composition, the biscuits of both groups were comparable: 23.8 g carbohydrate, 4.4 g protein and 11.8 g fat, which in total yielded 215 kcal per 36 g biscuit. On a weekly basis, schoolchildren in the experimental group would receive ~326.3 μg retinol activity equivalents (RAEs) for vitamin A per day, which satisfied 59.3% of the Recommended Dietary Allowance (RDA) of vitamin A for children aged 7–12 years old (Institute of Medicine, 2001). The personnel who conducted the biscuit distribution and sample processing was blinded to the group assignments, and the blinding procedure was done by a third party that was not involved in the study. Subjects who did not complete at least 70% of intervention or both, were excluded from the analysis.

2.3. Examination of soil-transmitted helminths

The schoolchildren were given a food-grade wrapping paper and a labelled wide mouth with screw-cap containers a day before faecal sample collection. They were advised to defaecate directly into the containers or pass the faeces onto the food-grade wrapping paper and then transfer it into the containers immediately using the scoop attached to the lid of the containers. Any faecal samples that were found contaminated with urine were excluded. Fresh faecal samples were collected at baseline before the administration of anthelmintic treatments, 3- and 6-month after interventions. The samples were examined via Kato-Katz and formalin-ether concentration techniques for the presence of STHs (T. trichiura, A. lumbricoides and hookworm) eggs (Cheesbrough, 1999; Martin and Beaver, 1968). The results of Kato-Katz techniques were recorded as eggs per gram (epg) of faecal sample. The intensity of infection was categorised as heavy, moderate, or light according to classifications proposed by the World Health Organization (2002).

2.4. Ethical consideration

The study was carried out in compliance with the principles laid down in the Declaration of Helsinki. Ethical approval for this study was obtained from the Medical Research Ethics Committee, Ministry of Health (MOH) Malaysia (NMRR No: NMRR-16–1905-32547 [IIR]). Assent forms were signed by the schoolchildren and written informed consents were attained from all the literate parents before the study commencement. As for illiterate guardians, verbal consents with their thumbprints on the informed consent form were obtained, with the procedure being witnessed and formally recorded. Permissions to conduct the study were obtained from the Department of Orang Asli Development (JAKOA), MOH and the Ministry of Education (MOE). The study was also registered at ClinicalTrials.gov (Identifier: NCT03256123).

2.5. Statistical analysis

Statistical analysis was conducted based on the principle of per-protocol analysis. RStudio (R version 4.1.2, Vienna, Austria) was used for the following statistical analyses. The categorical data were presented as numbers and percentages. A study conducted among the indigenous communities of Malaysia reported 100% cure rates of triple-dose albendazole treatment against A. lumbricoides and hookworm, while the cure rate for trichuriasis was recorded at 64.6% (Tee et al., 2022). In view of the high efficacy of triple-dose albendazole treatment against A. lumbricodes and hookworm, the reinfection rates of these two STH species were calculated using the following formula based on the assumption that most of the infections were cured after triple-dose albendazole administration: (Number of infected subjects at 3- or 6-month of intervention / number of infected subjects before anthelmintic treatment) x 100% (Al-Mekhlafi et al., 2014). With regards to T. trichiura, the term reinfection rate was not used because of low efficacy of albendazole against T. trichiura, but the prevalence of T. trichiura infection at 3- or 6-month of intervention with respect to baseline level will be calculated based on the same formula to ease interpretation. The exact confidence intervals (95% two-sided) for reinfection rates or prevalence with respect to baseline level of STHs were calculated using the Clopper-Pearson method (Newcombe, 1998). Chi-square test and Fisher’s exact test were applied to compare the reinfection rates or the prevalence with respect to baseline level between the experimental and control groups. The distribution of continuous variables was examined for normality using the Kolmogorov-Smirnov test before analysis. It was suggested that arithmetic mean is a more biologically justifiable measure to illustrate STH egg counts, in preference to the median or geometric mean (Al-Mekhlafi et al., 2014; Montresor, 2007). Therefore, although the egg counts were found to be not normally distributed, they were presented as arithmetic mean and 95% confidence interval (95% CI). The STH epg values were introduced as the dependent variable in linear mixed-effects model (LMM) fitted by restricted maximum likelihood estimation method (REML), using function lme from package nlme, to evaluate the effects of 3- and 6-month intervention on the intensities of each STH species after anthelmintic treatment. Intervention group, timepoint, and their interaction term (group x timepoint), as well as state and school type were included in the model as fixed effects, with participant as a random effect. The statistical significance of group x timepoint interaction term was estimated by Type III analysis of variance (ANOVA) via function anova.lme from package nlme. Pairwise comparisons of STH epg values were performed with function emmeans from the emmeans package with P value adjustment using Bonferroni corrections and reported for each timepoint per group. A P value <0.05 was considered statistically significant.

3. Results

3.1. Baseline characteristics of participants

A total of 651 students were enrolled in the current study after the exclusion of 513 students (44.1%) who missed the screening assessment or were not meeting the inclusion criteria. The schematic flow of the participation of subjects for the present study is shown in Figure 1. A total of 343 schoolchildren completed follow-up and were included in the analysis: 95 schoolchildren neither received complete deworming course nor allocated intervention due to absenteeism, 211 schoolchildren were lost during the follow-up period due to absenteeism, and 2 schoolchildren in experimental group were excluded from analysis as they did not complete 70% of intervention. Baseline characteristics of the subjects are presented in Table 1. Overall, there were 152 boys (44.3%) and 191 girls (55.7%) in the population with a mean age of 9.9 ± 1.3 years old. The distribution of age and sex were similar across groups. More than half of the study population (66.5%) were Orang Asli schoolchildren, who are the indigenous minority peoples of Peninsular Malaysia as depicted in our previous published data (Tan et al., 2022). A majority (64.2%) were from <RM500 monthly household income families. Overall, 90 subjects (26.2%) were diagnosed with confirmed VAD, while 253 (73.8%) were found to have marginal VAD, and there was no significant difference in the prevalence of VAD (experimental group= 26.1% confirmed VAD and 73.9% marginal VAD, and control group = 26.3% confirmed VAD and 73.7% marginal VAD) between the two groups.

Fig. 1.

Fig. 1.

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Participants flow diagram. The administration of triple-dose albendazole (one dose of 400 mg for three consecutive days) was conducted at baseline before the initiation of intervention. The follow-ups of intervention were conducted every 3 months for a duration of 6 months.

Table 1.

Baseline characteristics of the schoolchildren (N=343).

Characteristics Experimental group (N=153) Control group (N=190) Total (N=343)
Socio-demographic characteristics
Age (years) 9.9 ± 1.3 9.9 ± 1.4 9.9 ± 1.3
Sex
 Boy 64 (41.8) 88 (46.3) 152 (44.3)
 Girl 89 (58.2) 102 (53.7) 191 (55.7)
State
 Pahang 48 (31.4) 66 (34.7) 114 (33.2)
 Perak 53 (34.6) 46 (24.2) 99 (28.9)
 Johor 15 (9.8) 13 (6.8) 28 (8.2)
 Sabah 20 (13.1) 31 (16.3) 51 (14.9)
 Sarawak 17 (11.1) 34 (17.9) 51 (14.9)
School Type
 Orang Aslia Schools 116 (75.8) 112 (58.9) 228 (66.5)
 Non-Orang Asli Schools 37 (24.2) 78 (41.1) 115 (33.5)
Household income
 <RM500 101 (69.2) 96 (59.6) 197 (64.2)
 ≥RM500 45 (30.8) 65 (40.4) 110 (35.8)
Biochemical indicators
VAD status
 Confirmed (Retinol <0.70 μmol/L) 40 (26.1) 50 (26.3) 90 (26.2)
 Marginal (Retinol 0.70 - <1.05 μmol/L) 113 (73.9) 140 (73.7) 253 (73.8)
Anaemia (Hb <115 g/L) 26 (17.2) 31 (16.6) 57 (16.9)
Iron deficiency (Ferritin <15 μg/L) 19 (12.7) 33 (17.6) 52 (15.4)
Iron-deficiency anaemia (Hb <115 g/L and ferritin <15 μg/L) 14 (9.5) 10 (5.3) 24 (7.2)
STH infection
T. trichiura
 Prevalence 103 (67.3) 108 (56.8) 211 (61.5)
 Intensity
  Heavy 14 (9.2) 13 (6.8) 27 (7.9)
  Moderate 49 (32.0) 55 (28.9) 104 (30.3)
  Light 40 (26.1) 40 (21.1) 80 (23.3)
A. lumbricoides
 Prevalence 51 (33.3) 40 (21.1) 91 (26.5)
 Intensity
  Heavy 10 (6.5) 3 (1.6) 13 (3.8)
  Moderate 22 (14.4) 15 (7.9) 37 (10.8)
  Light 19 (12.4) 22 (11.6) 41 (12.0)
Hookworm
 Prevalenceb 39 (25.5) 37 (19.5) 76 (22.2)
Infected with at least one type of STH 111 (72.5) 114 (60.0) 225 (65.6)
Infected with only one type of STH 44 (28.8) 59 (31.1) 103 (30.0)
 Only T. trichiura 36 (23.5) 53 (27.9) 89 (25.9)
 Only A. lumbricoides 2 (1.3) 2 (1.1) 4 (1.2)
 Only hookworm 6 (3.9) 4 (2.1) 10 (2.9)
Co-infection of STHs 67 (43.8) 55 (28.9) 122 (35.6)
 T. trichiura + A. lumbricoides 34 (22.2) 22 (11.6) 56 (16.3)
 T. trichiura + hookworm 18 (11.8) 17 (8.9) 35 (10.2)
 T. trichiura + A. lumbricoides + hookworm 15 (9.8) 16 (8.4) 31 (9.0)
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All values are given as n (%).

VAD, Vitamin A deficiency; Hb, haemoglobin; STH, soil-transmitted helminths

a

Indigenous minority peoples of Peninsular Malaysia

b

All subjects were infected with light intensity of hookworm

3.2. Baseline prevalence and intensity of STH infection

Overall, the prevalence of STH infection was 65.5%, with T. trichiura (61.5%) being the most common STH species, followed by A. lumbricoides (26.5%) and hookworm (22.2%) (Table 1). The experimental group exhibited significantly higher prevalence of A. lumbricoides (33.3%) compared to the control group (21.1%) (P=0.02), while there is no significant difference between the groups for T. trichiura and hookworm infections (Table 2).

Table 2.

Reinfection rates or prevalence of STHs with respect to the baseline level and at 3-month and 6-month of intervention (N=343).

Experimental group (n=153) Control group (n=190) P value
T. trichiura
Baseline 67.3 (59.3, 74.7) 56.8 (49.5, 64.0) 0.06
3-montha 79.6 (70.5, 86.9) 77.8 (68.8, 85.2) 0.88
6-montha 79.6 (70.5, 86.9) 83.3 (74.9, 89.8) 0.60
A. lumbricoides
Baseline 33.3 (25.9, 41.4) 21.1 (15.5, 27.5) 0.01
3-montha 5.9 (1.2, 16.2) 17.5 (7.3, 32.8) 0.10b
6-montha 35.3 (22.4, 49.9) 60.0 (43.3, 75.1) 0.03
Hookworm
Baseline 25.5 (18.8, 33.2) 19.5 (14.1, 25.8) 0.23
3-montha 5.1 (0.6, 17.3) 8.1 (1.7, 21.9) 0.67b
6-montha 10.3 (2.9, 24.2) 10.8 (3.0, 25.4) 1.00b
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All values are prevalence or reinfection rates expressed as percentage (95% confidence interval).

a

Reinfection rate or prevalence of STHs with respect to baseline level (95% confidence interval) at 3-month or 6-month of intervention: (Reinfection rates / prevalence of STHs with respect to baseline level = Number of infected subjects at 3-month or 6-month of intervention/ number of infected subjects before deworming treatment x 100), Chi-square test.

b

Fisher’s exact test.

Among those infected with T. trichiura, 12.8% and almost half of them (49.3%) had heavy and moderate infections, respectively (Table 1). Similarly, 14.3% and 40.7% of the A. lumbricoides-infected schoolchildren were found to have heavy and moderate infections, respectively. All hookworm infections in this population were of light intensity. Based on Table 3, at baseline, the intensity (arithmetic mean epg) of A. lumbricoides is significantly higher in the experimental group (8968.00, 95% CI [5334.94, 12601.06]) compared to the control group (2963.77, 95% CI [1417.00, 4510.54]) (P<0.001), while there were no significant differences between the groups for the intensities of T. trichiura and hookworm.

Table 3.

Intensities of STH reinfections after 3-month and 6-month of intervention (N=343).

Experimental group (n=153) Control group (n=190) P value (Group x timepoint)a P value (Between group)b P value (Relative to baseline within
experimental group)c 		P value (Relative to baseline within
control group)d
T. trichuris 0.36
Baseline 2996.83 (2043.41, 3950.26) 2972.23 (1801.98, 4142.48) 1.00
3-month 1089.00 (633.18, 1544.82) 665.82 (260.91, 1070.73) 1.00 0.001 < 0.001
6-month 1592.67 (681.42, 2503.91) 639.24 (290.60, 987.89) 1.00 0.05 < 0.001
A. lumbricoides < 0.001
Baseline 8968.00 (5334.94, 12601.06) 2963.77 (1417.00, 4510.54) <0.001
3-month 1.67 (−0.56, 3.89) 79.72 (−75.15, 234.59) 1.00 < 0.001 0.54
6-month 1818.33 (464.14, 3172.53) 3567.32 (513.07, 6621.56) 1.00 < 0.001 1.00
Hookworm 0.81
Baseline 45.83 (15.99, 75.68) 41.20 (17.60, 64.81) 1.00
3-month 2.17 (−1.80, 6.13) 0.54 (−0.11, 1.18) 1.00 0.004 0.002
6-month 1.33 (−0.40, 3.07) 6.71 (−1.44, 14.86) 1.00 0.003 0.02
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All values are arithmetic mean counts of eggs per gram (epg) of faecal sample with 95% confidence interval, adjusted for state and school type.

a

Estimated by type III analysis of variance (ANOVA) on the linear mixed model.

b

Pairwise comparisons with P value adjustment using Bonferroni corrections to evaluate the difference in arithmetic mean epg between group within the same timepoint.

c

Pairwise comparisons with P value adjustment using Bonferroni corrections to evaluate the changes in arithmetic mean epg from baseline to 3-month of intervention within the same group.

d

Pairwise comparisons with P value adjustment using Bonferroni corrections to evaluate the changes in arithmetic mean epg from baseline to 6-month of intervention within the same group.

More than one-third (35.6%) of the schoolchildren had mixed infections with ascariasis and trichuriasis (16.3%) being the most prevalent coinfection, followed by 10.2% of the children infected with both T. trichuris and hookworm, and 9.0% infected with three types of STHs (A. lumbricoides, T. trichiura and hookworm) (Table 1). The experimental group had almost 2 times higher coinfection of T. trichiura and A. lumbricoides (22.2%) compared to the control group (11.6%).

3.3. Effects of RPO-enriched biscuit supplementation on STH reinfection rates

The effects of the intervention on the reinfection rates of A. lumbricoides and hookworm, as well as the prevalence of T. trichiura with respect to its baseline prevalence and at 3- and 6-month of intervention are illustrated in Table 2. At 3-month, the overall prevalence of T. trichiura, A. lumbricoides and hookworm reached 78.7%, 11.0% and 6.6%, respectively of the baseline levels. The reinfection rate of A. lumbricoides was lower in the experimental group (5.9%) compared with control group (17.5%), but statistically insignificant. Similar reinfection rate of hookworm was recorded in both groups (experimental group = 5.1% and control group = 8.1%). Likewise, the prevalence of T. trichiura at 3-month of intervention with respect to its baseline prevalence was similar across experimental (79.6%) and control (77.8%) groups.

At 6-month, the overall reinfection rates of A. lumbricoides and hookworm were recorded at 10.5% and 46.2%, respectively; while the prevalence of T. trichiura was 81.5%, with respect to the baseline prevalence. On the other hand, we observed that the reinfection rates of A. lumbricoides reached almost half of the baseline levels. Interestingly, the reinfection rate of A. lumbricoides in the control group (60.0%) was almost two-fold higher than in the experimental group (35.3%) (P=0.03). There was similar increase in the reinfection rate of hookworm between experimental (10.3%) and control (10.8%) groups. Similarly, there was no significant difference between groups for T. trichiura infection at 6-month with respect to the baseline prevalence.

3.4. Effects of RPO-enriched biscuits supplementation on the intensities of STH reinfection

The changes in the intensities of STH reinfection after 3- and 6-month of interventions are demonstrated in Table 3. A significant interaction of group and timepoint was only observed in the intensity of A. lumbricoides (P<0.001), which indicates that the changes in A. lumbricoides intensities across timepoints were significantly different between groups. Based on post-hoc analysis, significant reductions in the intensities of A. lumbricoides at 3-month from baseline intensities were only observed in experimental group (P<0.001). At 6-month of intervention, we observed an apparent reinfection of A. lumbricoides in the control group, whereby the intensity (3567.77, 95% CI [513.07, 6621.56]) was not significantly different from the intensities at baseline (2963.77, 95% CI [1417.00, 4510.54]). In contrast, the intensity of A. lumbricoides in the experimental group at this point (1818.33, 95% CI [464.14, 3172.53]) was only 20.3% of the baseline intensity (8968.00, 95% CI [5334.94, 12601.06]) (P<0.001). On the other hand, there were significant reductions in the intensities of T. trichiura and hookworm at 3- and 6-month from the baseline intensities, with no significant difference observed between groups.

4. Discussion

The findings of this study illustrated the infection intensity of all three species of STH at 3-month were significantly reduced from the baseline findings (P<0.001), but similar across both the experimental and control groups. These observations indicate the reductions in the intensities of STHs among the schoolchildren at 3-month were mainly because of triple-dose albendazole treatment, rather than the effects of supplementation. In general, a re-assessment of STH infections at 14 to 21 days after anthelmintic treatment is conducted to determine the efficacy of treatment (Horton, 2000). However, the evaluation of anthelmintic treatment efficacy at 14 to 21 days was unable to be carried out due to manpower and logistic constraints, resulting in the study being conducted at a prolonged period of 3-month in this study. Similar outcomes were reported by Al-Mekhlafi et al. (2014), where the intensities of all STH species at 3-month were significantly lower than the respective baseline intensities, but no significant difference between vitamin A and placebo groups was observed. Besides, the overall reinfection rates or the prevalence with respect to the baseline prevalence at 3-month of intervention were high and similar between experimental and control groups, but varied by STH species. Based on a systematic review and meta-analysis conducted by Jia et al. (2012), the reinfection rates of T. trichiura, A. lumbricoides and hookworm at 3-month after treatment were recorded at 36%, 26% and 30%, respectively, with the exclusions of studies with low adherence (loss to follow-up rate > 30%), low baseline prevalence (< 10%) and poor cure rates (< 20% for T. trichiura, < 80% for A. lumbricoides and < 20% for hookworm). At 3-month of intervention, the overall reinfection rates of STHs demonstrated in the present study were considerably low for A. lumbricoides (11.0%) and hookworm (6.6%); while for T. trichiura, the prevalence with respect to its baseline level (78.7%) was considered high. The variations in the reinfection rates of STHs observed could be due to the differences in endemicity level, intervention involved, age, sex, hygiene and behavioural conditions, socioeconomic status, and nutritional status in different populations (Al-Mekhlafi et al., 2008; Jia et al., 2012; Yap et al., 2013). In addition, the high prevalence of T. trichiura with respect to its baseline level observed in the current study could be due to the poor cure rate or low efficacy of albendazole against T. trichiura (Adegnika et al., 2015; Steinmann et al., 2011). A high reinfection rate of T. trichiura was demonstrated by Yap et al. (2013), where 79.8% of the schoolchildren of ethnic minority background in Yunnan province, China were found to be still infected with T. trichiura at 3-month after triple-dose albendazole treatment, with cure rate at 1-month recorded at only 19.6%.

Interestingly, we observed a significantly lower reinfection rate of A. lumbricoides among schoolchildren supplemented with RPO-enriched biscuits (35.3%) as compared to the control group (60.0%) at 6-month of intervention. In the context of infection intensity of A. lumbricoides, there was apparent reinfection in the control group, while the reinfection of A. lumbricoides in the experimental group only achieved 8.8% of the baseline intensity. In a study conducted among preschool children in rural Panama, within 6 to 9 months after deworming, the mean epg of A. lumbricoides returned to pre-treatment levels (Halpenny et al., 2013). Likewise, previous study conducted among aboriginal children in rural Malaysia had demonstrated that the reinfection rate and infection intensity of A. lumbricoides were comparable to pre-treatment levels by 6 months after triple-dose albendazole treatment (Al-Mekhlafi et al., 2008). However, in this study, a similar reinfection pattern was not observed in the experimental group at 6-month, which suggests that RPO-enriched biscuit supplementation may potentially exhibit beneficial effects against the reinfection of A. lumbricoides.

Resistance against helminth infections was likely to be associated with T helper 2 (Th2)-mediated responses (humoral responses) (Dold and Holland, 2011). Besides, Turner et al. (2003) suggested that Th2-mediated responses have a protective role in reducing the intensity of A. lumbricoides infection. Jackson et al. (2004) further demonstrated that A. lumbricoides- and/or T. trichiura-infected individuals with higher susceptibility to helminthic infection were characterised with weaker Th2 responses, and those with higher resistance to reinfection at 8 to 9 months after anthelmintic were found to have higher pre-treatment interleukin-5 (IL-5) levels. Carotenoids are known to play an important role in modulating T-cell proliferation (Chew and Park, 2004). In this study, significant improvements in both blood α- and β-carotene levels were observed in RPO-enriched biscuits-supplemented group, but not in control group (unpublished data). Hence, the beneficial effects of RPO-enriched biscuit supplementation on the reinfection rate of A. lumbricoides could be due to the augmented immune responses owing to the high carotenoid content in RPO-enriched biscuits. Enhanced levels of Th and T inducer lymphocytes were also corroborated in human adults administrated with oral β-carotene supplementation (Alexander et al., 1985; Watson et al., 1991). Besides, the role of CD4+ T cells in the expulsion of STHs had been demonstrated in an animal model, where the expulsion of T. muris (a murine-specific counterpart to T. trichiura), was inhibited in the CD4+ T cell-depleted mice (Koyama et al., 1995). It has been previously reported that the ratio of CD4+ to CD8+ cells increased in healthy male volunteers after 9 months of β-carotene supplementation (Murata et al., 1994), which further suggests the potential immunomodulatory effects of carotenoid-rich RPO-enriched biscuits. The immune-modulating effects of carotenoids are known to be manifested via their metabolism to vitamin A and their subsequent involvements in the retinoic acid receptor (RAR)/retinoid-X receptor (RXR)-response pathways (Rühl, 2007).

Furthermore, the other potential mechanism underlying the beneficial effects of RPO-enriched biscuit supplementation on the reinfection rate of A. lumbricoides may be the enhanced integrity of mucosal epithelium. It was previously demonstrated that Trichinella spiralis and Nippostrongylus brasiliensi were surrounded by mucus preceding their expulsion, which infers the importance of the mucus barrier in protecting the host and facilitating parasite expulsion (Sharpe et al., 2018). Based on the findings of an in vitro study reported by Cheng et al. (2021), β-carotene may play a prominent role in reducing inflammation and improving intestinal barrier integrity, which was shown by the enhanced levels of tight junction proteins, the protein multiplexes that are essential for the intestinal mucosal barrier function. Low serum carotenoid concentrations were also found to be associated with impaired intestinal barrier integrity, which postulates that carotenoids may play an important role in ameliorating disturbed intestinal barrier function (Vieira et al., 2008). Taken all together, the findings of the present study suggest that carotenoids-rich RPO-enriched biscuit supplementation can potentially promote the re-establishment of intestinal barrier integrity, which may further protect the host from subsequent reinfection.

Comparing with previous studies that investigate the effects of vitamin A supplementation in combination with deworming treatment on STH reinfection, the administration of high dose vitamin A was either carried out once at baseline or every 2 months depending on the age groups (Al-Mekhlafi et al., 2014; Long et al., 2007; Payne et al., 2007). Long term supplementation of high dose vitamin A was associated with hypervitaminosis A or vitamin A toxicity, which adversely affects health conditions (Blomhoff, 2001). However, high intakes of carotenoids do not cause hypervitaminosis A as the conversion of carotenoids is affected by vitamin A status (Haskell, 2012). Hence, the supplementation of carotenoid-rich RPO-enriched biscuit in the current study could serve as an alternative long-term supplementation in conjunction of deworming treatment to control STH reinfection.

It is also worth mentioning that the overall reinfection rate of A. lumbricoides (46.2%) and hookworm (10.5%) at 6-month were considerably low compared to the findings reported in other studies conducted among Orang Asli schoolchildren, where the reinfection rates of A. lumbricoides and hookworm at 6 months after deworming treatment were noticeably high at about 75% and 50% of the pre-treatment levels, respectively (Al-Mekhlafi et al., 2014; Al-Mekhlafi et al., 2008). Poor knowledge, attitude, and practices of Orang Asli in rural Malaysia towards STH infections were previously reported in a survey conducted by Nasr et al. (2013). The effectiveness of school-based health education on parasitic infections among Orang Asli schoolchildren in Malaysia was demonstrated by Al-Delaimy et al. (2014), where a significant reduction in the intensity of T. trichiura, A. lumbricoides and hookworm, as well as a decrease in prevalence of hookworm infections were observed among those who received the health education for a duration of 3 months. Prior to the conduct of the intervention, there was a briefing session with the schoolchildren together with their parents/guardians to discuss their blood biochemical and STH infections assessments results during the screening phase. In addition to that, they were being educated on the health consequences of STH infections and how to control STH infections by practising good personal hygiene. Hence, the briefing sessions may have created awareness among the study populations about STH infections, which further helped in reducing the overall reinfection rates of STHs among the schoolchildren.

To date, this is the first study to report the supplementation of RPO-enriched biscuits on STH reinfection, hence the outcomes of the current study shed some light on the new integrated approach to control STH reinfections. Besides, the longitudinal design of this study gives additional strength to the study to examine the patterns of STH reinfections over a duration of 6 months after initiation of intervention. It is also notable to mention that the conduct of this study created awareness of STH infections among the schoolchildren as reflected by the comparatively lower overall reinfection rates of A. lumbricoides and hookworm infections at 6-month if compared to the previous studies conducted in Orang Asli communities. In consideration of the STH reinfections observed among the schoolchildren, these children were given another dose of deworming medicine at 6-month after the completion of the study, as a gesture of goodwill. Nevertheless, there are several limitations in the current study. Owing to the lack of manpower as well as logistic and time constraint as the interventions were conducted concurrently in ten schools located in five different states, the assessment of STH infection at 14 to 21 days was unable to be carried out. However, the complete deworming of A. lumbricoides and hookworm after the triple-dose albendazole treatment was postulated and the assessments of reinfection at 3-month and 6-month were justifiable in view of the high efficacy of triple-dose albendazole treatment against these 2 STH species (Sungkar et al., 2019; Tee et al., 2022). The clinical evidence on the immunomodulating effects of carotenoids supplementation were found to be limited and outdated, which most of the data were published in ‘90s. Therefore, more studies are needed to provide more holistic and up-to-date evidence. Also, the inclusion of immunological markers in future studies would be valuable to elucidate the mechanistic action of carotenoid-rich RPO-enriched on the host immune system in controlling STH reinfection.

In conclusion, the findings of the present study suggest that RPO-enriched biscuit supplementation may have beneficial effects on A. lumbricoides reinfection at 6-month of intervention after deworming treatment among schoolchildren with vitamin A deficiency in rural areas of Malaysia. We have argued that these effects may be attributable to the carotenoids that are abundantly found in RPO which play an important role in enhancing immune response and mucosal epithelium integrity. However, more studies are warranted to confirm whether RPO supplementation could produce the same parasite-specific beneficial effects in other community settings, as well as to understand the mechanisms involved.

Highlights.

  • Red palm olein (RPO) is the richest plant source of carotenoids.

  • Effects of RPO biscuit supplementation on STH reinfection after deworming was studied.

  • RPO biscuit group had significantly lower reinfection rate of A. lumbricoides.

  • RPO biscuit group had significant reduction in infection intensity of A. lumbricoides.

Acknowledgements

First and foremost, we would like to express our deepest appreciations to all the schoolchildren and their parents for their voluntary participation in this study. Sincere acknowledgements to the Ministry of Education (MOE), Ministry of Health (MOH), and Department of Orang Asli Development (JAKOA), the head of Orang Asli villages (Tok Batin), school principals and teachers for the permissions given to carry out sample and data collections from the schoolchildren. We are also grateful to Carotino Sdn Bhd., Noraini’s Cookies Worldwide Sdn. Bhd., medical doctors, medical assistances, nurses, MPOB staffs, and internship students for their assistance and support throughout the study. The study was supported by the RMK-11 (Eleventh Malaysia Plan) Grant (PD219/16), which managed by Malaysian Palm Oil Board. This research was supported (in part) by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (NIAID).

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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