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RESEARCH Open Access

Investigations into the association between soil-transmitted helminth infections, haemoglobin and child development indices in Manufahi District, Timor-Leste Suzy J. Campbell1*, Susana V. Nery1, Catherine A. D’Este1, Darren J. Gray1,2,3, James S. McCarthy3,4, Rebecca J. Traub5, Ross M. Andrews6, Stacey Llewellyn4, Andrew J. Vallely7, Gail M. Williams3 and Archie C. A. Clements1

Abstract

Background: Timor-Leste has a high prevalence of soil-transmitted helminth (STH) infections. High proportions of the population have been reported as being anaemic, and extremely high proportions of children as stunted or wasted.

There have been no published analyses of the contributions of STH to these morbidity outcomes in Timor-Leste.

Methods: Using baseline cross-sectional data from 24 communities (18 communities enrolled in a cluster randomised controlled trial, and identically-collected data from six additional communities), analyses of the association between STH infections and community haemoglobin and child development indices were undertaken. Stool samples were assessed for STH using qPCR and participant haemoglobin, heights and weights were measured. Questionnaires were administered to collect demographic and socioeconomic data. Intensity of infection was categorised using correlational analysis between qPCR quantification cycle values and eggs per gram of faeces equivalents, with algorithms generated from seeding experiments. Mixed-effects logistic and multinomial regression were used to assess the association between STH infection intensity classes and anaemia, and child stunting, wasting and underweight.

Results: Very high stunting (60%), underweight (60%), and wasting (20%) in children, but low anaemia prevalence (15%), were found in the study communities. STH were not significantly associated with morbidity outcomes. Male children and those in the poorest socioeconomic quintile were significantly more likely to be moderately and severely stunted. Male children were significantly more likely than female children to be severely underweight. Increasing age was also a risk factor for being underweight. Few risk factors emerged for wasting in these analyses.

Conclusions: According to World Health Organization international reference standards, levels of child morbidity in this population constitute a public health emergency, although the international reference standards need to be critically evaluated for their applicability in Timor-Leste. Strategies to improve child development and morbidity outcomes, for example via nutrition and iron supplementation programmes, are recommended for these communities. Despite the apparent lack of an association from STH in driving anaemia, stunting, wasting and underweight, high endemicity suggests a need for STH control strategies.

Trial registration: Australian and New Zealand Clinical Trials Registry ACTRN12614000680662; retrospectively registered.

Keywords: Soil-transmitted helminths, Necator americanus, Ascaris, Morbidity, Anaemia, Stunting, Wasting, PCR

* Correspondence: suzanne.campbell@lstmed.ac.uk 1Research School of Population Health, College of Medicine, Biology and Environment, The Australian National University, 62 Mills Rd, Acton, ACT 2601, Australia Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Campbell et al. Parasites & Vectors (2017) 10:192 DOI 10.1186/s13071-017-2084-x http://crossmark.crossref.org/dialog/?doi=10.1186/s13071-017-2084-x&domain=pdf https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12614000680662 mailto:suzanne.campbell@lstmed.ac.uk http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/publicdomain/zero/1.0/

Background Southeast Asia harbours one-third of the world’s soil-transmitted helminths (STH) [1], and Timor-Leste is one of the poorest countries in the region [2]. Two recent cross-sectional studies identified moderate school-based (29%) [3] and high community-based (69%) [4] STH prevalence in the Manufahi District of Timor-Leste, using different diagnostic techniques. Spe-cifically, in this area, prevalence of Necator americanus was 60%, Ascaris spp. 24%, Ancylostoma spp. 4.7%, and Trichuris trichiura 0.33%, with Giardia duodenalis the most common protozoan identified (13%) [4]. Inad-equate water and sanitation infrastructure and hygiene behaviours in this area likely contribute to high STH endemicity [4], which in turn could contribute signifi-cantly to morbidity.

STH have previously been associated with anaemia, stunting and wasting [5–9]. The mechanism whereby hookworms contribute to reduced haemoglobin and more indirectly to poor growth and development outcomes is via blood loss and inflammation, with heavily-infected people at greatest risk of morbidity [10].

Ascaris lumbricoides is not considered a contributor to blood loss (reviewed in [11]) and T. trichiura contributes to blood loss in heavy infection. Whilst STH have been shown to be associated with stunting and wasting [12, 13] the causal relationship is not clear.

Despite inability to establish causality with observa-tional analyses, investigating the relationship between STH infection and haemoglobin concentration, and child anthropometric indices, is of considerable import-ance in Timor-Leste. Extremely high proportions of Timorese under 5 years of age are reported as stunted (50%) and wasted (11%) [14], yet knowledge of the contribution of STH to this is very limited, with no prior investigations identified. A cross-sectional survey in 2008 found 22% of children aged 24–59 months of age were anaemic [15]. Additionally, a demographic health survey in 2009–2010 found 38% of Timorese children aged 6–59 months, and 21% of Timorese women aged 15–49 years were anaemic [16]. Since this time, risk factors for anaemia in women of reproductive age have been investigated [17]. However, limited data on STH have prevented STH contributions from being assessed.

Using quantitative polymerase chain reaction (qPCR) for STH diagnosis and intensity of infection assessment [18], we aim to (i) determine classes of STH infection in-tensity from PCR-derived data, and (ii) provide the first analysis of morbidity associated with STH infections in Manufahi District, Timor-Leste. We used an algorithm to correlate quantification cycle (Cq) values from qPCR to eggs per gram of faeces (epg) equivalents, determined by seeding experiments [18]. The association between in-tensity of N. americanus and Ascaris spp. (as exposures) and mean haemoglobin concentrations and anaemia diagnosis (as outcomes) were then investigated for all community members, and associations with stunting, underweight and low BMI-for-age (as a measure of wasting) as outcomes in children aged one to 18 years were also investigated.

Methods Study setting, design and collection of data This study was conducted as one of a series of baseline analyses for the “WASH for Worms” RCT, which aims to determine the extent of a reduction in burden of STH by integrating mass chemotherapy and community-based water, sanitation and hygiene (WASH) pro-grammes [19]. Rural communities in Manufahi District were selected for the study according to RCT-related inclusion and exclusion criteria (including being identi-fied by the Timorese government as high-priority communities for WASH interventions) [19]. The RCT commenced in May 2012, with baseline surveys conducted in 18 communities until October 2013.

Identically-collected data from six communities in Manufahi District were added; these communities were enrolled at the same time as the RCT communities but were not randomly allocated to each trial arm. Full details of study area and design [19], questionnaires and parasitological diagnostic approaches [4, 18] are pro-vided elsewhere. Briefly, Manufahi District is comprised of rural Timorese villages with subsistence-based liveli-hoods. Community consultations and consent elicitation were conducted before questionnaire administration.

Children aged less than 12 months and pregnant women in the first trimester of pregnancy were excluded be-cause they could not receive albendazole. Questionnaires were used to record details of water, sanitation and hygiene (WASH) practices, household and individual socioeconomic characteristics [4].

Measurement of anaemia status and anthropometry Anaemia status was measured for all ages with haemoglobin concentration assessed by finger-prick blood test using a portable haemoglobinometer (HemoCue, Ängelholm, Sweden). Haemoglobin concentrations can be used to assess anaemia (being inadequate intakes and reserves of host iron and protein [20]). Data on haemoglobin were linked to household GPS coordi-nates and adjusted by -2 grams per litre for elevation of 1000 m above sea level in accordance with World Health Organization (WHO) recommendations [21];

data from four communities, and part of a fifth community, were adjusted in this way; no communities had an elevation above 1500 m. Haemoglobin was initially classified based on WHO definitions of anaemia severity (Table 1 [21]); however due to small numbers was re-

Campbell et al. Parasites & Vectors (2017) 10:192 Page 2 of 15 categorised as a binary variable (anaemic/non-anaemic);

which was used as the primary outcome.

For children aged two to < 18 years, weight was measured to the nearest 0.1 kg using electronic scales (CAMRY, ED-301), and height was measured to the nearest 0.1 cm using a portable stadiometer (Wedderburn, WSHRP). Children aged 1 to 2 years had length measured supine with a measuring mat (Wedderburn, SE210), and weight measured by taring (i.e. with the child held by an adult, and the adult’s weight subsequently deducted).

Height-for-age (HAZ) and BMI-for-age (BMIZ, i.e.

weight over height2-for-age) z-scores were calculated for children aged 1– < 18 years. Weight-for-age (WAZ) z-scores were calculated for children aged 1– < 10 years, standardised to the international 2006 reference popula-tion using the software WHO Anthro and Anthroplus, for children up to five and aged five and over, respect-ively [22, 23]. These scores are expressed as differences from the reference median and are calculated based on sex and date of birth of each individual. Age in days was used for z-score calculations. Children of uncertain birthdate were assigned a mid-year birthdate (15th June) and records followed up with parents subsequently;

1038 children’s records (95.4%) had completed birthdates. WAZ is only calculated up to 10 years of age, because it is considered inadequate for monitoring growth beyond this age [24]; BMIZ complements HAZ in the assessment of thinness (low BMIZ) [24], and was used instead of weight-for-height (which is calculated for under-fives only) to assess wasting. Each of these continuous outcomes was categorised, with individuals classified as moderately stunted, underweight or wasted if HAZ, WAZ and BMIZ respectively were more than two standard deviations below the reference median, and severely stunted, underweight or wasted, respect-ively, if the z-scores were more than three standard deviations below the reference median [25].

Assessment of STH infection Single stool samples were collected, preserved in 5% potassium dichromate at room temperature, transported to QIMR Berghofer Medical Research Institute, Brisbane, Australia, and tested by multiplex qPCR for presence and intensity of STH and protozoal infection using a method previously described [18]. Prevalence of all STH were assessed in this way. Based on highest prevalences, N. americanus and Ascaris spp. (reported at genus level) were analysed for intensity of infection. For purposes of this analysis, qPCR quantification cycle (Cq) values, representing the amplification cycle where the signal exceeded background, was interpolated as a meas-ure of the parasite DNA load in the stool sample [26], using a validated internal control. Cq-values were expressed on a log10 scale of the linear equation with fluorescence (i.e. log(b0 + b1x)), where the slope (b1) and the y-intercept (b0) are provided from the PCR output and x is the Cq-value. Lower values therefore denote heavier intensity infection. In these assays a Cq-value of 31 for Ascaris, and a Cq-value of 35 for N. americanus, were set as the limits for detection of infection [18]. All assays showed Cq-values for the internal control within the expected range.

Data analysis Data were analysed in STATA 14.0 (Stata Corporation, College Station, Texas). A wealth quintile was con-structed using principal components analysis of variables assessing ownership of household assets (including animals, transport and appliances), house floor type, reported income and presence of electricity as reported previously [4], according to established methods [27].

For faecal specimens, two runs were taken for each

PCR assay. The arithmetic mean of the two (untrans-formed) Cq-values was taken to create a single measure per person. For ease of interpretation and for comparison with other studies, untransformed Cq-values were categorised in these analyses. Receiver-operating characteristic curves (ROCs) were used to assign initial cut-points for Cq-values, using a gener-ated morbidity score (see Additional file 1). However, a very weak relationship between intensity of infection for either N. americanus or Ascaris and the morbidity score was observed (see below). This led to extremely poor predictive capacity using ROCs, so this tech-nique was not ultimately used. Full detail of this is reported in Additional file 1, as statistical assignment of categories to infection intensities represent an important contribution that may be useful in

Table 1 Definitions of anaemia used in this study, measured as grams per litre (source [23])

No anaemia Mild anaemia Moderate anaemia Severe anaemia

Children < 5 years ≥ 110 100–109 70–99 < 70

Children 5–11 years ≥ 115 110–114 80–109 < 80

Children 12–14 years ≥ 120 110–119 80–109 < 80

Non-pregnant women (≥ 15 years) ≥ 120 110–119 80–109 < 80

Pregnant women ≥ 110 100–109 70–99 < 70

Men (≥ 15 years) ≥ 130 110–129 80–109 < 80

Campbell et al. Parasites & Vectors (2017) 10:192 Page 3 of 15 assigning qPCR data to intensity of infection cat-egories elsewhere.

An algorithm to assign intensity of infection based on approximations of epg was used for this analysis, with intensity classes being based on those endorsed by the WHO to represent high, moderate and low-intensity infections [28] (see Additional file 1 for detail). This algorithm was generated from seeding experiments as previously described [18] and was based on the linear re-lationship between the log10 of epg and Cq-value [18].

Because WHO endorsed categorisations of epg intensity are based on the Kato-Katz diagnostic technique [28], a recovery factor of 0.2 was applied to current epg classes of infection intensity (based on a 20% recovery rate determined for faecal flotation of Ascaris eggs (R. Traub, unpublished data). This recovery factor was used in the absence of a recovery factor being known for Kato-Katz and the known poor accuracy of this technique in diag-nosing STH infections [29]) (Table 2).

Using classes of infection intensity for epg based on inter-national standards [28], the Cq cut-point that correlated with heavy-intensity infection of (≥ 4000*5 = 20,000) epg was selected for N. americanus, and the Cq cut-point that correlated with heavy-intensity infection of (≥ 50,000*5 = 250,000) epg was selected for Ascaris.

The final intensity of infection variables for both STH were therefore categorised according to heavy-intensity, moderate- to low-intensity (hereafter called “moderate-intensity”), and no infection, whereby moderate-intensity infection was all Cq between the heavy-infection cut-point and the detectable Cq limits of 31 for Ascaris, and 35 for N. americanus. Sensitiv-ity analyses were then undertaken, comparing these cut-points to a model that applied a cut-point of 10% heavy-intensity infection (based on the percentage of endemic populations deemed by the WHO as likely to suffer morbidity from heavy infections [28]), a model of 15% heavy-intensity infection, a model based on quintiles of infection intensity, and also comparing results from applying cut-points that have been reported elsewhere [30–32] to these data. The intensity cut-points selected using the WHO endorsed thresholds as estimated by the algorithm performed comparably to these other cut-points on sensitivity analyses based on parameter esti-mates and Akaike’s Information Criteria, and thus were used in all further analyses.

Chi-square tests were conducted to compare preva-lence of morbidity by age, sex and socioeconomic quintile; these variables were retained as core variables in all multivariable models. The associations between intensity of infection and morbidity by age group and sex were also explored. Mixed-effects logistic regression (for binary-coded anaemia as outcome) and mixed-effects multinomial regression within a generalised structural equation model framework (separately for outcomes of stunting, wasting and underweight) were undertaken to account for correlation among outcomes at the household and village level.

Initially univariable analyses were undertaken with each

STH species, G. duodenalis, age group (categorical), sex as a binary variable, and socioeconomic quintile (categorical) included as explanatory variables for each of the morbidity outcomes. Although there was a high prevalence of N.

americanus in this population [4], there was no multicolli-nearity with other STH species. Variables with P < 0.2 from univariable analyses were added stepwise into a base model which included age group, sex, socioeconomic quintile, the categorical intensity of infection explanatory variables for N. americanus and Ascaris (described above), and binary Ancylostoma infection, until the most parsimonious adjusted model for each outcome was achieved. Variables were retained in final models if P < 0.1 on the Wald test.

For models of stunting, underweight and wasting, anaemia was additionally included as a core variable be-cause of its importance as a potential confounder. There were insufficient observations to investigate anaemia risk factors in children under five or within other childhood age groups. Anaemia was therefore modelled separately for children (aged 1– < 18 years) and adults, given the difference in prevalence and potential risk factors for these two groups. There were additionally insufficient observations to investigate stunting, wasting or under-weight by age groups in regression analyses. Prevalence of both STH and morbidity vary with age and sex, and both the literature [33] and our earlier analyses [4] indicated the potential for moderation of the STH-morbidity relationship by age and sex. Given this, and observed differences in relationships between classes of N. americanus infection intensity and stunting across age and sex (identified in cross-tabulations), interactions between sex and N. americanus intensity of infection, and age group and N. americanus intensity of infection, were investigated. Interactions were investigated by generating models with and without the interaction term and comparing these using the likelihood ratio test, with

Table 2 Ascaris spp. and Necator americanus intensity of infection quantification cycle (Cq) cut-points between heavy and moderate morbidity

Soil-transmitted helminth

Eggs per gram of faeces (epg) classa

EPG class with recovery factor appliedb

Corresponding Cq-value c

N. americanus ≥ 4000 20,000 24.6

Ascaris spp. ≥ 50,000 250,000 15.4

Abbreviations: Cq, quantification cycle; epg, eggs per gram of faeces aEggs per gram of faeces intensity classes follow WHO definitions [28] bRecovery factor of 0.2 applied to epg intensity class based on recovery factor determined from faecal flotation (R. Traub, unpublished data) cDNA intensity from exponentiated Cq-values

Campbell et al. Parasites & Vectors (2017) 10:192 Page 4 of 15 a requirement for P < 0.1 for likelihood ratio tests, for interaction inclusion in the model. Using these criteria, no interactions were required in adjusted models for an-aemia. A sex by N. americanus intensity of infection interaction was retained in the adjusted child stunting model, and an age group by N. americanus intensity of infection interaction was retained in the adjusted child underweight model. For the multinomial child anthro-pometry models, sensitivity analyses using binary-coded prevalence of outcome (of main effects only, i.e. no interaction terms) were undertaken to investigate the impact of increasing power. Additionally, post-hoc cal-culations were performed to determine the power to de-tect effects within each outcome, adjusting for correlations within households and villages. These calcu-lations indicated 80% power, with a 5% significance level, to detect odds ratios of 3.3 or more for anaemia out-comes, relative risks of 1.4 to 1.7 for stunting and under-weight outcomes (depending on level of severity), and (reflecting lower numbers) relative risks of 2.1 to 9.5 for wasting as an outcome (according to level of severity).

Results Prevalence of morbidity Respondents from communities who provided both a stool and finger-prick blood sample were included in analyses of haemoglobin (2038 participants). Only 15% of the population suffered from anaemia, with the ma-jority of these (11%) being only mildly anaemic, and only three children being severely anaemic (Tables 3 and 4).

Anaemia was most prevalent in younger ages, and gen-erally decreased with increasing age (P < 0.0001) (Fig. 1).

The observed zero prevalence of anaemia in females aged 65 years and over is of interest, although participant numbers in older age groups were generally low. Adult women of reproductive age (aged 18– < 45 years) had higher prevalence of anaemia than men of the same age (18.0 vs 7.9%, P < 0.0001). The overall prevalence of N. americanus and Ascaris were 61, and 24%, respectively [4]. The prevalence of Ascaris was highest amongst children of preschool age, whereas N.

americanus was most prevalent in adults (Table 3) [4].

Children 1– < 18 years old who provided stool and had height and/or weight measured were included in ana-lyses of z-scores (Table 4). Extremely high levels of stunting (60%), underweight (60%), and wasting (20%) were found, with 25% being severely stunted, 20% se-verely underweight, and 4.5% severely wasted (Table 4).

This morbidity is reflected in the mean z-scores for each measure, all of which are well below zero. Stunting, underweight and wasting varied by age group with, gen-erally, greater proportions of older children (≥ 10 years) experiencing severe morbidity compared to younger age groups. The prevalence of stunting was significantly higher among poorer households compared to wealthier households (P < 0.0001) and among males compared to females (P < 0.0001), but the overall association between stunting and age was non-significant (P = 0.117).

Exploratory analyses indicated some unexpected, but not statistically significant, trends. A greater proportion of uninfected males were severely stunted than N.

americanus-infected males (Table 5). This trend did not exist for females, who instead showed greater proportions of uninfected having normal (i.e. non-stunted) growth. Similarly, children aged 1 to 5 years with N. americanus infection generally had lower prevalence of severe stunting; a trend that reversed in the oldest age group (where a lower proportion of severe stunting was seen in uninfected children (Table 5). These complex and varying underlying relationships confirmed our decision to investigate interaction terms using regression models.

Prevalence and severity of being underweight was moderately higher for males (P = 0.004), and generally increased by age (P = 0.002), but not socioeconomic quintile (P = 0.088). Prevalence and severity of wasting increased by age (P < 0.0001), but did not differ by sex (P = 0.651) or socioeconomic quintile (P = 0.666).

Assignment of DNA intensity cut-points From PCR output, there were no Cq-values above 35, indicating good reproducibility of the assays. There were very weak relationships between STH infection and all morbidity outcomes, which hampered statis-tical assignment of cut-points using ROC curves.

Table 2 shows the final selected cut-points. Using our cut-points, 1155 (52%) of study subjects were categorised as having heavy-intensity N. americanus

Table 3 Baseline characteristics of study participants (N = 2038)

Baseline community characteristics

All ages (N = 2038)

Children; aged 1– < 18 years (N = 1018)

Adults; aged ≥ 18 years (N = 1020) n (%) n (%) n (%)

Mean haemoglobin (g/L, (SD))

131 (16)a 126 (13)a 136 (16)a

Non-anaemic 1 731 (85) 872 (86) 859 (84)

Mildly anaemic 222 (11) 95 (9.3) 127 (13)

Moderately/severely anaemicb

86 (4.2) 51 (5.0) 33 (3.3)

Ascaris spp. prevalence 501 (25) 302 (30) 199 (20)

N. americanus prevalence 1 238 (61) 522 (51) 716 (70)

Ancylostoma spp. prevalence 99 (4.9) 44 (4.3) 55 (5.4)

G. duodenalis prevalence 261 (13) 203 (20) 58 (5.7)

Abbreviations: g/l grams per litre; SD standard deviation aMean and standard deviation presented instead of n and % bModerate and severe anaemia categories combined to maintain participant confidentiality

Campbell et al. Parasites & Vectors (2017) 10:192 Page 5 of 15 infection, and 191 (8.6%) moderate-intensity infection (Table 6). For Ascaris, 220 (9.9%) people had heavy-intensity infection, and 318 (14%) people moderate-intensity infection. Amongst infected people, N.

americanus mean infection intensity was Cq 21.5 (95% confidence interval, CI: 21.3–21.8), and for Ascaris 19.5 (95% CI: 19.2–19.9) (Table 6). Intensity of infection changed over age, with most heavy-intensity Ascaris infection occurring in young children. Heavy-intensity N. americanus infections were more evenly distributed across age groups, including older age groups.

Factors associated with anaemia Neither Ascaris nor N. americanus infection intensity were significantly associated with anaemia in the multi-variable models, although Ascaris moderate-intensity infection was marginally non-significant as a risk factor for adults (Table 7). Heavy N. americanus infection in children had a protective association with anaemia. How-ever, this was not a significant factor in adjusted models, potentially indicating the confounding effect of other model factors. Increasing age was a highly significant, strongly protective factor in univariable and adjusted models for

Table 4 Anthropometric characteristics of study participants (N = 2038)

Z-score characteristics

Height-for-age (HAZ)a All children 1– < 18 years (N = 983) n (%)

1– < 5 years (N = 267) n (%)

5– < 10 years (N = 365) n (%)

≥ 10 years (N = 351) n (%)

Mean HAZ (SD)a -2.25 (1.22)a

Not stunted 391 (40) 105 (39) 164 (45) 122 (35)

Moderately stunted 347 (35) 89 (33) 132 (36) 126 (36)

Severely stunted 245 (25) 73 (27) 69 (19) 103 (29)

Weight-for-age (WAZ)b All children 1– < 10 years (N = 639) n (%)

1– < 5 years (N = 268) n (%)

5– < 10 years (N = 371) n (%)

Mean WAZ (SD)b -2.19 (1.03)a

Not underweight 257 (40) 124 (46) 133 (36)

Moderately underweight 253 (40) 108 (40) 145 (39)

Severely underweight 129 (20) 36 (13) 93 (25)

BMI-for-age (BMIZ)a All children 1– < 18 years (N = 985) n (%)

1– < 5 years (N = 266) n (%)

5– < 10 years (N = 369) n (%)

≥ 10 years (N = 350) n (%)

Mean BMIZ (SD)a -1.19 (1.03)a

Not wasted 796 (81) 242 (91) 308 (84) 246 (70)

Moderately wasted 145 (15) 20 (7.5) 47 (13) 78 (22)

Severely wasted 44 (4.5) 4 (1.5) 14 (3.8) 26 (7.4)

Abbreviations: HAZ height-for-age, WAZ weight-for-age, BMIZ BMI-for-age, SD standard deviation aHAZ (stunting) and BMI (wasting) calculated for individuals 12 months to < 18 years of age bWAZ (as indicator of underweight) only calculated for individuals 12 months to 10 years of age

Fig. 1 Anaemia distribution by sex and age group (n = 2000)

Campbell et al. Parasites & Vectors (2017) 10:192 Page 6 of 15 children. There was no sex difference in odds of anaemia in children. Children in the poorest socioeconomic quintile had twice the odds of anaemia relative to those in the wealthiest quintile. For adults, age was non-significant, and neither sex nor socioeconomic status were associated with anaemia, with no evidence of an overall trend in odds with decreasing socioeconomic quintile.

Factors associated with stunting No level of either N. americanus or Ascaris infection intensity was associated with stunting of any severity in this population (Table 8). However, whilst not sta-tistically significant, heavy-intensity Ascaris infection was associated with higher relative risks for both moderate and severe stunting; these relative risks were of reasonable size (moderate stunting adjusted relative risk (ARR) 1.6, 95% CI: 0.87–2.9; severe stunting ARR 2.0, 95% CI: 0.87–4.4). Sensitivity analysis of stunting prevalence showed that heavy-intensity Ascaris infection was marginally non-significantly associated with greater odds of stunting compared to uninfected children (adjusted odds ratio (AOR) 1.8, 95% CI: 0.98–3.4, P = 0.057; results not shown). In the multinomial stunting model (Table 8), Ancylostoma infection was associated with moderate and severe stunting. However, the effect was in the opposite direction to anticipated: children with Ancy-lostoma infection were significantly less likely to have moderate or severe stunting than uninfected children.

Due to inclusion of a sex by N. americanus inter-action term, we report results of the association between N. americanus and stunting separately for males and females. Females with N. americanus infec-tion of any intensity had no significant association with stunting of any severity. Being male and having heavy-intensity N. americanus infection (relative to being male and having no N. americanus infection) was associated with significantly reduced risk of severe stunting. It is important to note, however, that the main effect of N. americanus infection intensity showed that there was no association with stunting in females (the reference category). The main effect for sex indicates that, in those with no N. americanus in-fection, being male was highly significantly associated with almost three times the risk of moderate stunting, Table 5 Baseline characteristics of children by category of stunting (N = 592)

Baseline community characteristics

Not stunted n (%)

Moderately stunted n (%)

Severely stunted n (%)

Males

High N. americanus infection intensity

92 (36) 96 (37) 70 (27)

Moderate N. americanus infection intensity

10 (31) 10 (31) 12 (38)

No N. americanus infection 57 (27) 80 (38) 76 (36)

Total 159 (32) 186 (37) 158 (31)

Females

High N. americanus infection intensity

80 (48) 54 (32) 35 (21)

Moderate N. americanus infection intensity

16 (39) 17 (41) 8 (20)

No N. americanus infection 136 (50) 90 (33) 44 (16)

Total 232 (48) 161 (34) 87 (18)

Age group 1 to 5 years

High N. americanus infection intensity

38 (48) 27 (34) 14 (18)

Moderate N. americanus infection intensity

12 (43) 11 (39) 5 (18)

No N. americanus infection 89 (38) 79 (34) 65 (28)

Total 139 (41) 117 (34) 84 (25)

Age group 6 to 11 years

High N. americanus infection intensity

94 (43) 79 (36) 47 (21)

Moderate N. americanus infection intensity

10 (35) 11 (38) 8 (28)

No N. americanus infection 78 (43) 62 (34) 41 (23)

Total 182 (42) 152 (35) 96 (22)

Age group 12 to 17 years

High N. americanus infection intensity

40 (31) 44 (34) 44 (34)

Moderate N. americanus infection intensity

4 (25) 5 (31) 7 (44)

No N. americanus infection 26 (38) 29 (42) 14 (20)

Total 70 (33) 78 (37) 65 (31)

Table 6 Necator americanus and Ascaris spp. intensity of infection profile. Numbers indicate all those who provided a stool sample, and therefore are not consistent with the numbers used in separate morbidity analyses

STH Infection intensity profile n (%) Mean Cq (95% CI)

Heavy intensity Moderate to low intensity No infection

N. americanus 1155 (52.0) 191 (8.6) 873 (39.0) 21.5 (21.3–21.8)

Ascaris spp. 220 (9.9) 310 (14.0) 1689 (76.0) 19.5 (19.2–19.9)

Campbell et al. Parasites & Vectors (2017) 10:192 Page 7 of 15 and almost seven times the risk of severe stunting.

Relative to children aged one to five, children aged 12 to 17 had almost twice the risk of moderate stunting, and almost three times the risk of severe stunting, although no significant associations were seen in children aged 6 to 11 years. Children in the poorest socioeconomic quintile had twice the risk of moderate stunting, and five times the risk of severe stunting, compared to children in the wealthiest socioeconomic quintile. Anaemia was not a risk factor for stunting.

Factors associated with being underweight The association between underweight and N. ameri-canus infection is reported separately by age group be-cause of the age group by N. americanus interaction term. In those aged one to five, N. americanus infection of any severity was not associated with being under-weight. Whilst this same association was evident for children aged six to ten with N. americanus infection, the association with being severely underweight was only marginally non-significant for those having moderate N.

americanus intensity of infection. Compared to children aged 1 to 5 years, being aged 6 to 10 years was signifi-cantly associated with three times the risk of being severely underweight, but no increased risk of being moderately underweight, in N. americanus-uninfected children. There was no association between intensity of Ascaris infection and being underweight in this popula-tion (Table 9). Ancylostoma infection was close to sig-nificance for being moderately underweight, but not for being severely underweight. The presence of G.

duodenalis infection, or of anaemia, were also not significantly associated with being underweight. Being male was highly significantly associated with three times greater risk of being severely underweight com-pared to normal growth children, but this association was not evident for moderate levels of underweight.

Household socioeconomic quintile was not associated with being underweight in these analyses, although the association with poorest socioeconomic quintile was only marginally not significant for being moderately underweight. With binary-coded underweight as out-come, a sensitivity analysis confirmed the consistency of results with the multinomial results.

Factors associated with wasting Although no intestinal parasites were associated with wasting in adjusted analyses, observation numbers in this model were very low for assessment of some categories (Table 10). Additionally, the presence of

Table 7 Odds ratios for anaemia, by Necator americanus and Ascaris spp. infection intensity, Manufahi District, Timor-Leste. Logistic regression was used for investigating factors associated with anaemia. The outcome variable is binomial anaemic/non-anaemic and associations therefore presented as odds ratios

Children; aged 1 < 18 years (N = 1018) Adults; aged≥ 18 years (N = 1020)

Univariable Multivariable Univariable Multivariable

Parameter OR 95% CI AOR 95% CI P OR 95% CI AOR 95% CI P

Ascaris heavy-intensity 0.93 0.49–1.8 0.91 0.46–1.8 0.8820 1.7 0.84–3.3 1.8 0.86–3.6 0.1165

Ascaris moderate-intensity 0.73 0.39–1.4 0.85 0.44–1.6 1.7** 1.04–2.9 1.6 0.93–2.7

N. americanus heavy-intensity 0.52*** 0.33–0.83 0.69 0.41–1.1 0.1606 0.92 0.62–1.4 0.86 0.56–1.3 0.7280

N. americanus moderate-intensity 0.42a 0.18–1.0 0.50a 0.21–1.2 0.99 0.52–1.9 1.0 0.52–1.9

Ancylostoma infection 1.5a 0.58–3.9 1.5a 0.58–4.1 0.3940 0.93a 0.42–2.1 0.68a 0.27–1.7 0.4130

G. duodenalis infection 0.92 0.56–1.5 0.42a 0.16–1.1

Male sex 1.1 0.70–1.6 0.97 0.64–1.5 0.8940 0.93 0.66–1.3 0.98 0.68–1.4 0.9180

Age group 6 to 11 years 0.35*** 0.22–0.54 0.39 0.24–0.61 < 0.0001

Age group 12 to 17 years 0.20*** 0.11–0.38 0.19 0.09–0.38 < 0.0001

Age group 65+ years 1.2 0.74–1.9 1.2 0.76–2.0 0.3920

Socioeconomic quintile 4 1.0 0.49–2.0 0.93 0.46–1.9 0.0098 0.58 0.31–1.1 0.58 0.31–1.1 0.3718

Socioeconomic quintile 3 0.52 0.23–1.2 0.49 0.22–1.1 0.85 0.48–1.5 0.83 0.46–1.5

Socioeconomic quintile 2 1.2 0.60–2.4 1.1 0.55–2.2 1.1 0.65–2.0 1.1 0.62–1.9

Socioeconomic quintile 1 (poorest) 2.1** 1.0–4.1 2.1 1.0–4.3 0.97 0.54–1.7 0.91 0.50–1.7

Notes: Ascaris (categorical infection intensity), N. americanus (categorical infection intensity), Ancylostoma infection prevalence (binary), age group, sex and socioeconomic quintile were included in all multivariable regression models as exposure outcomes and covariates. Reference categories: no Ascaris infection, no N. americanus infection, no Ancylostoma infection, no Giardia infection, age group 1–5 years (for child model), age group 18–64 years (for adult model), male sex, socioeconomic quintile 5 (wealthiest) Abbreviations: OR odds ratio, AOR adjusted odds ratio, CI confidence interval; P, Wald test P-value **P < 0.05, ***P < 0.01 in univariable analysis aLess than 10 observations in a subgroup; result should be interpreted cautiously Ascaris and N. americanus intensity infections were defined according to following cut-points: Ascaris: heavy-intensity Cq ≤ 15.4, moderate-intensity Cq > 15.4 < 31, no infection Cq ≥ 31; N. americanus: heavy-intensity Cq ≤ 24.6, moderate-intensity Cq > 24.6 < 35, no infection Cq ≥ 35

Campbell et al. Parasites & Vectors (2017) 10:192 Page 8 of 15 anaemia was not associated with wasting. Relative to being aged 1 to 5 years, being aged 6 to 11 years was associated with highly significant, threefold increased risk for being either moderately or severely wasted. Strikingly, this trend worsened amongst children aged 12 to 17 years, with four times the risk of moderate wasting, and seven times the risk of severe wasting, seen in these children relative to the youngest age group. There was no association between sex or socioeconomic status and categories of wasting.

Given low numbers with this multinomial wasting out-come, a sensitivity analysis was conducted using wasting as a binary-coded outcome; results were consistent with the multinomial analyses, with age group the sole significant factor.

Discussion In this first investigation of STH associations with haemoglobin and child development indices in Manufahi

District, Timor-Leste, a generally lower prevalence of anaemia than results reported previously [17] was observed, with 11% prevalence in children aged 1– < 5 years, and 18% prevalence in reproductive-aged women (18– < 45 years of age). For women, anaemia whilst moder-ately lower than reported previously [17], was signifi-cantly more frequent than in males of the same age, confirming the serious disease burden in this popula-tion group. This likely reflects the well-reported impact of pregnancies and menstruation on iron stores [34]. An-aemia in mothers is itself a risk factor for stunting or wast-ing in offspring. Iron-folic acid supplementation for pregnant women has been implemented by the Ministry of Health across all districts of Timor-Leste since 2003, with 61% of pregnant women reporting taking supple-ments in 2009–2010 [16].

Despite the difference in prevalence, being female was not demonstrated to be a risk factor for anaemia in

Table 8 Relative risk ratios for stunting, Manufahi District, Timor-Leste

Moderate stunting (n = 347) Severe stunting (n = 245)

Parameter RR 95% CI ARR 95% CI P RR 95% CI ARR 95% CI P

Ascaris heavy-intensity 1.5 0.83–2.7 1.6 0.87–2.9 0.3007 1.8 0.79–4.0 2.0 0.87–4.4 0.2002

Ascaris moderate-intensity 1.1 0.65–1.8 1.0 0.61–1.7 1.0 0.50–2.0 0.93 0.46–1.9

N. americanus heavy-intensitya 0.82 0.56–1.2 0.82 0.47–1.4 0.6140 0.73 0.43–1.2 0.91 0.41–2.0 0.8647

N. americanus moderate-intensitya 0.86 0.42–1.7 1.3 0.52–3.1 0.67 0.27–1.7 0.70 0.18–2.7

Male sexb 2.0*** 1.4–2.8 2.8 1.7–4.7 < 0.0001 3.7*** 2.3–6.0 6.8 3.4–13.7 < 0.0001

N. americanus heavy-intensity infection in malesc

0.65 0.31–1.4 0.2540 0.31 0.11–0.85 0.0230

N. americanus moderate-intensity infection in malesc

0.43 0.10–1.8 0.2440 0.82 0.13–5.3 0.8340

Ancylostoma infection 0.26***d 0.11–0.65 0.27d 0.11–0.68 0.0050 0.33**d 0.11–0.99 0.37d 0.12–1.1 0.0780

Giardia infection 1.5 0.96–2.3 1.1 0.61–2.0

Anaemia prevalence 1.2 0.68–2.0 1.1 0.65–2.0 0.6600 1.7 0.86–3.3 1.7 0.83–3.4 0.1470

Age group 6 to 11 years 0.92 0.63–1.4 1.1 0.71–1.6 0.7090 0.72 0.43–1.2 1.1 0.60–1.9 0.8620

Age group 12 to 17 years 1.4 0.86–2.3 1.7 0.98–2.8 0.0580 1.7 0.87–3.2 2.7 1.3–5.4 0.0060

Socioeconomic quintile 4 1.1 0.59–2.1 1.2 0.63–2.2 0.2415 0.91 0.34–2.5 1.1 0.39–2.9 0.0165

Socioeconomic quintile 3 1.4 0.69–2.7 1.4 0.70–2.8 1.2 0.41–3.4 1.3 0.45–3.8

Socioeconomic quintile 2 1.3 0.65–2.4 1.2 0.64–2.3 1.5 0.55–4.0 1.5 0.57–4.2

Socioeconomic quintile 1 (poorest) 2.3** 1.1–4.6 2.3 1.1–4.6 5.1*** 1.8–14.2 5.1 1.8–14.7

Notes: RRs in bold had univariable P< 0.2 and were entered in multivariable regression models; for correct interpretation of this table, if a variable was significant for moderate stunting but not severe stunting, it was still included, therefore on occasion moderate stunting adjusted RRs are significant when severe stunting adjusted RRs are not, and vice versa. Ascaris (categorical infection intensity), N. americanus (categorical infection intensity), Ancylostoma infection prevalence (binary), anaemia prevalence (binary), age group, sex and socioeconomic quintile were included in all multivariable regression models as exposure outcomes and covariates. A sex*N. americanus interaction is included in the model. Reference categories: no Ascaris infection, no N. americanus infection, female sex, no N. americanus infection in males (multivariable only), no Ancylostoma infection, no Giardia infection, no anaemia, age group 1–5 years, socioeconomic quintile 5 (wealthiest) Abbreviations: RR relative risk, ARR adjusted relative risk, CI confidence interval; P, Wald test P-value **P < 0.05, ***P < 0.01 in univariable analysis aThe N. americanus main effect, being N. americanus infection intensity relative to no N. americanus infection in females (because females are the reference group) bThe male sex main effect, being the relative risk of male sex relative to female sex when there is no N. americanus infection (reference group) cN. americanus infection intensity in males is relative to no N. americanus infection in males (because males and moderate- or heavy-intensity N. americanus infection are not the reference groups) dTen observations or less in subgroup; result should be interpreted cautiously Normal growth (no stunting) is reference category, i.e. moderate and severe stunting need to be interpreted relative to this reference. Ascaris and N. americanus intensity infections defined according to following cut-points: Ascaris: heavy-intensity Cq≤ 15.4, moderate-intensity Cq > 15.4 < 31, no infection Cq≥ 31; N. americanus: heavy-intensity Cq≤ 24.6, moderate-intensity Cq > 24.6 < 35, no infection Cq≥ 35

Campbell et al. Parasites & Vectors (2017) 10:192 Page 9 of 15 adults or children. Interestingly, neither N. americanus nor Ascaris of either class of infection intensity were significant risk factors, this despite the well-recognised association between hookworm infection and blood loss.

Whilst N. americanus is implicated in blood loss, it causes measurably less blood loss than Ancylostoma duodenale [10]. Blood loss due to parasite infection needs to be greater than nutritional reserves and required intake for anaemia to develop [35]. In this population the prevalence of the more pathogenic hook-worm species Ancylostoma duodenale was very low, representing a possible explanation for the weak associ-ation between STH (particularly hookworm) and anaemia. Similar negligible associations have been iden-tified in N. americanus-endemic populations elsewhere [35]. Socioeconomic status and age were not important risk factors for anaemia in adults, but were important, highly significant, risk factors for children. Different risk factor associations between children and adults point to the need to conduct further age-stratified analyses, ideally with children aged less than five analysed separ-ately due to the higher prevalence of anaemia in this age group; observation numbers limited further age-stratification in our analysis. Anaemia can be caused by multiple concurrent factors including inadequate dietary iron, and it is inherently difficult to control for all of these in epidemiological studies. The lack of other iden-tified risk factors in our models would suggest that additional unmeasured factors may be influencing these results. Of note, the prevalence of malaria had dramatic-ally declined in Timor-Leste prior to the commence-ment of this study [36], indicating that this is not a likely confounding factor.

In the study area, an extremely high prevalence of stunt-ing, underweight and wasting in children were observed compared to the international reference population, with considerable proportions of severe stunting, underweight and wasting. These are higher than national estimates, possibly reflecting the rurality of the study communities.

Our reported prevalence, whilst being at a district, not

Table 9 Relative risk ratios for being underweight, Manufahi District, Timor-Leste

Moderate underweight (n = 253) Severe underweight (n = 129)

Parameter RR 95% CI ARR 95% CI P RR 95% CI ARR 95% CI P

Ascaris heavy-intensity 1.0 0.53–2.1 0.85 0.41–1.7 0.8925 1.6 0.72–3.5 1.2 0.49–2.8 0.9379

Ascaris moderate-intensity 1.1 0.60–2.1 1.0 0.52–1.9 1.5 0.73–3.3 1.0 0.46–2.3

N. americanus heavy-intensitya 1.0 0.64–1.7 0.59 0.28–1.2 0.3584 0.85 0.47–1.5 0.66 0.25–1.8 0.1519

N. americanus moderate-intensitya 0.94 0.41–2.2 0.76 0.26–2.2 0.87d 0.31–2.5 0.12d 0.01–1.2

Age group 6 to 10 yearsb 1.4 0.91–2.2 1.2 0.63–2.2 0.5930 2.8*** 1.6–4.8 3.3 1.5–7.0 0.0030

Age group 6 to 10 years and N.

americanus heavy-intensity infectionc

1.4 0.80–2.5 1.8 0.67–4.8 0.2510 1.7 0.81–3.6 0.70 0.20–2.4 0.5740

Age group 6 to 10 years and N.

americanus moderate-intensity infectionc

1.8 0.39–8.2 2.1 0.30–13.9 0.4630 7.0 1.36–35.8 15.6 0.91–266.4 0.0580

Ancylostoma infection 2.5 0.88–6.8 2.8 0.94–8.1 0.0640 0.69d 0.15–3.2 0.86d 0.18–4.2 0.8480

Giardia infection 0.97 0.58–1.6 1.1 0.58–2.1

Anaemia prevalence 0.99 0.56–1.8 0.98 0.54–1.8 0.9430 0.70 0.34–1.5 0.85 0.39–1.9 0.6820

Male sex 1.3 0.85–2.0 1.3 0.81–2.0 0.3040 2.4*** 1.4–4.2 3.1 1.7–5.6 < 0.0001

Socioeconomic quintile 4 1.4 0.66–3.1 1.5 0.66–3.3 0.2606 0.57 0.20–1.6 0.66 0.23–1.9 0.3095

Socioeconomic quintile 3 1.1 0.46–2.4 1.1 0.48–2.6 1.0 0.36–2.8 1.1 0.37–3.0

Socioeconomic quintile 2 1.1 0.53–2.5 1.2 0.54–2.7 0.87 0.33–2.3 0.96 0.36–2.6

Socioeconomic quintile 1 (poorest) 2.4 1.0–5.6 2.5 1.0–6.1 2.0 0.72–5.8 2.2 0.76–6.6

Notes: Ascaris (categorical infection intensity), N. americanus (categorical infection intensity), Ancylostoma infection prevalence (binary), anaemia prevalence (binary), age group, sex and socioeconomic quintile were included in all multivariable regression models as exposure outcomes and covariates. Being underweight not measured in children aged 11 to 17 years. An age group*N. americanus interaction is included in the model. Reference categories: No Ascaris infection, no N. americanus infection, age group 1–5 years, no N. americanus infection in age group 6 to 10 years (multivariable only), no Ancylostoma infection, no Giardia infection, no anaemia, female sex, socioeconomic quintile 5 (wealthiest) Abbreviations: RR relative risk, ARR adjusted relative risk, CI confidence interval, P, Wald test P-value **P < 0.05, ***P < 0.01 in univariable analysis aThe N. americanus main effect, being N. americanus infection intensity relative to no N. americanus infection in age group 1 to 5 years (reference age group) bThe age group 6 to 10 years main effect, being the relative risk of being aged 6 to 10 relative to being aged 1 to 5 when there is no N. americanus infection (reference group) cN. americanus infection intensity in age group 6 to 10 years is relative to no N. americanus infection in age group 6 to 10 years (because age group 6 to 10 years and moderate- or heavy-intensity N. americanus infection are not the reference groups) d10 observations or less in subgroup; result should be interpreted cautiously Normal weight is reference category, i.e. moderate and severe underweight need to be interpreted relative to this reference. Ascaris and N. americanus intensity infections defined according to following cut-points: Ascaris: heavy-intensity Cq ≤ 15.4, moderate-intensity Cq > 15.4 < 31, no infection Cq ≥ 31; N. americanus:

heavy-intensity Cq ≤ 24.6, moderate-intensity Cq > 24.6 < 35, no infection Cq ≥ 35

Campbell et al. Parasites & Vectors (2017) 10:192 Page 10 of 15 national level (and therefore perhaps more susceptible to small geographic area fluctuations), are amongst some of the highest reported rates in the world [37]. This is despite relatively low community prevalence of anaemia. Propor-tions of child wasting in particular are well above the 15% level of severity classified as critical [38]. Wasting repre-sents rapid and severe malnutrition such as starvation, although it can also be the result of chronic unfavourable conditions [38]. With strong links between wasting and child mortality [38] this level is considered to be a public health emergency [39] that requires immediate response.

However, as a cautionary note, the application of the 2006 WHO international reference standards to the Timorese population has not been assessed, and there is a possibility of this population being of a…