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Presence of Giardia lamblia in stools of six‐ to 18‐month old asymptomatic Malawians is associated with children's growth failure

Abstract Aim Despite high pathogen burden and malnutrition in low‐income settings, knowledge on relationship between asymptomatic viral or parasitic infections, nutrition and growth is insufficient. We studied these relationships in a cohort of six‐month‐old Malawian infants. Methods As part of a nutrient supplementation trial for 12 months, we documented disease symptoms of 840 participant daily and anthropometric measurements every three months. Stool specimens were collected every six months and analysed for Giardia lamblia, Cryptosporidium species and enterovirus, rotavirus, norovirus, parechovirus and rhinovirus using polymerase chain reaction (PCR). The prevalence of the microbes was compared to the children's linear growth and the dietary. Results The prevalence of the microbes was similar in every intervention group. All age groups combined, children negative for G. lamblia had a mean standard deviation (SD) of −0.01 (0.49) change in length‐for‐age Z‐score (LAZ), compared to −0.12 (0.045) among G. lamblia positive children (difference −0.10, 95% CI −0.21 to −0.00, p = 0.047). The LAZ change difference was also statistically significant (p = 0.042) at age of 18–21 months but not at the other time points. Conclusion Asymptomatic G. lamblia infection was mainly associated with growth reduction in certain three‐month periods. The result refers to the chronic nature of G. lamblia infection.


INTRODUCTION
Globally, linear growth failure or stunting affected an estimated 154.8 million, 22.9% of under five-year-old children in 2016 (1). Inadequate nutrition and intestinal infections are considered to be the main causes of childhood stunting (2) but results from antibiotic and probiotic interventions have been insufficient (3). In addition, Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study did not find diarrhoea to be a major risk factor for poor growth. However, the study identified asymptomatic enteropathogen burden as an important contributor to growth faltering in children (4).
Few studies have addressed the association of asymptomatic viral or parasitic infections and growth. We studied the epidemiology of two parasites and five viruses among asymptomatic Malawian children at six to 18 months of age and observed that primarily the asymptomatic Giardia lamblia infection was associated with subsequent growth reduction. To better assess the growth impact of non-chronic viral infections, more sensitive outcome variables are recommended with short follow-up periods. prevalence of enteric pathogens or their consequences without diarrhoea, particularly in young children, is still poorly understood (7). However, it has been suggested that acute infections impact the children's linear growth through a direct as enteric and indirect as respiratory pathways (8).
A primary aim of this study was to test whether the prevalence of G. lamblia, Cryptosporidium species, enterovirus, rotavirus, norovirus, parechovirus and rhinovirus, detected in faecal samples among asymptomatic Malawian children at age of six, 12 and 18 months, is associated with linear growth reduction in the subsequent three-month period. A secondary aim was to study if a dietary supplementation would have an impact on the prevalence of the studied microbes in the children's stools.

Study design and specimen collection
This was a prospective cohort study, nested in a clinical Lungwena Child Nutrition Intervention Study (LCNI-5) registration ID: NCT00524446, assessing the effect of dietary supplementation with lipid-based nutrient supplements (LNS) on early childhood growth (9). The trial was conducted in Lungwena and Malindi, in two rural Malawian communities. Potentially eligible participants were identified through community census in the study area and invited to an enrolment session, where infants were screened for eligibility. The inclusion criteria included age 5.50-6.50 months, residence in the study area and informed consent from an authorised guardian. The exclusion criteria were weight-for-length Z-score (WLZ) <80% of the World Health Organization (WHO) reference median or presence of oedema, severe illness warranting hospitalisation on the enrolment day, history of peanut allergy, concurrent participation in another clinical trial and any symptoms of food intolerance within 30 minutes after ingesting a five g test dose of LNS used in the trial (9).
The study population was enrolled between 28 January 2008 and 25 May 2010 and comprised of 840 six-month-old infants. After the enrolment, the participants were provided with one of the four dietary for 12 months. Dietary supplementation included either 54 g/day of milk or soy containing LNS, 72 g/day fortified maize-soy flour or in a control group, no extra food supplements.
The morbidity information was used to exclude those participants having illness symptoms at collection time. We collected data with picture calendar recorded by guardian. The calendar had separate columns for the following symptoms: fever, cough, diarrhoea and other. The recorded information was reviewed and cross-checked by fieldworkers at each two weekly food delivery visit for completeness. From the information we collected at enrolment, 31 children had had diarrhoea within 14 days prior to stool sample collection, 22 within seven days and 11 within two days. This information describes the data reported before the first (inclusion) sample was taken. Those participants who did not have the symptoms and were healthy in their guardians' views comprised our study subjects.
Faecal samples were collected at child age of six, 12 and 18 months to detect the prevalence of the pathogens. Guardians collected a faecal sample from the participants at each time point and brought the sample in a 30 mL stool container to the health centre. The time between defecation and faecal freezing was not more than six hours. Bacterial findings (10,11) and prevalence of selected viruses and parasites and their predictors (12) in Malawian children as part of the same nutritional trial have been reported earlier. Now, we report viral and parasitic results in relation to children's growth and the nutrition interventions.
The trial adhered to the principles of the Declaration of Helsinki and regulatory guidelines in Malawi. The research and ethics committee of the College of Medicine, University of Malawi and the ethical committee of the Pirkanmaa Hospital District, Finland reviewed and approved the trial protocol.

Sample preparations and real-time polymerase chain reaction (PCR) analysis
We isolated viruses and parasites using nucleic acids QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) from 10% stool suspension. We used real-time PCR to characterise enterovirus, rotavirus, norovirus, parechovirus and rhinovirus (13,14), and parasites G. lamblia and Cryptosporidium species (15). We considered a specimen as a positive sample if two or three of the triplicate PCR runs gave a positive test result. We excluded the data if the actual date of the stool sample was off by plus or minus four weeks from the prescheduled target date.

Anthropometric indices
Trained research assistants took anthropometric measurements at every three months from the child age of six to 21 months. Assistants measured length using a Kiddimetre length board (Raven Equipment Ltd., Essex, UK). They weighed the children using a SECA 735 electronic child weighing scale (Chasmors Ltd., London, UK). We calculated length-for-age Z-scores (LAZ) and WLZ using WHO Child Growth Standards (STATA igrowup package) (16). We excluded measurement if the actual measurement date was off by plus or minus four weeks from the target age.

Statistical analysis
For prevalence of viruses and parasites, we calculated percentages by intervention group as number of positive samples at each study visit. For LAZ and change in LAZ, we calculated group means and used least squares regression to estimate differences between groups. For all time points combined, we took intragroup correlation due to multiple measurements per participant into account by using robust standard errors for clustered data. We rejected a null hypothesis if two-sided p < 0.05. Prevalence of virus and parasite by intervention group is presented without covariate adjustment. Analysis of association between virus or parasite and LAZ and change in LAZ is adjusted for child's virus or parasite status at enrolment, maternal height, maternal body mass index at enrolment, child sex, maternal

RESULTS
Of the 1312 infants who were identified through community census, 472 were either ineligible, or they were not brought to an enrolment session ( Fig. 1). At six months of age, the mean standard deviation (SD) LAZ of the participants was À1.75 (0.97) and the mean WLZ was 0.46 (1.05). Less than five per cent of the participants had access to a piped water, and 91% of the participants had a sanitary facility, a pit latrine. There were no differences in the background characteristics between the intervention groups (Table 1). When all the time points and intervention groups were combined, the prevalence of tested microbes ranged from 2.0% of rotavirus to 75.9% of enterovirus. The percentage of positive stools for G. lamblia both at six and 12 months of age was 6.2%, the positivity both at 12 and 18 months of age was 10.6% and the positivity at six, 12 and 18 months of age was 5.1%. There was no statistically significant association between the dietary followed by the children and the prevalence of any of the studied microbes at any age (Table 2).
Because there were no differences in the prevalence of pathogens between the intervention groups, the subgroups were combined for the growth analyses. All age groups combined, children negative for G. lamblia had a mean SD of À0.01 (0.49) change in LAZ, whereas the figure was À0.12 (0.45) among those positive for G. lamblia (difference À0.10, 95% CI À0.21 to À0.00, p = 0.047). The LAZ change difference was largest and statistically significant (p = 0.042) between 18 and 21 months of age and smaller and statistically not significant at earlier time points. Children negative for rhinovirus at 12 months of age had a mean SD of À0.10 (0.51) change in LAZ, whereas the figure was À0.38 (0.50) among those positive for rhinovirus (difference À0.27, 95% CI À0.49 to À0.06, p = 0.011). With the exception of rhinovirus at this time point, the prevalence of other studied viruses was not associated with the children's linear growth in the subsequent three-month period (Table 3).
Generally, there was no association between the microbial prevalence at six, 12 or 18 months and attained LAZ by the same time point. The only exception was Cryptosporidium detection at 18 months of age when the mean (95% CI) LAZ was 0.87 (À1.64 to À0.10, p = 0.026) units lower among children with Cryptosporidium in their stool than those without it (Table 4).

DISCUSSION
The main aim of this study was to analyse if intestinal presence of G. lamblia, Cryptosporidium species, or  enterovirus, rotavirus, norovirus, parechovirus or rhinovirus in early childhood is associated with subsequent growth restriction among children living in Sub-Saharan resource-poor areas. A secondary aim was to assess if dietary supplementation with LNS or corn-soy flour would be associated with microbial detection. In a sample of 840 asymptomatic children aged six to 18 months, there was a high prevalence of viruses and parasites. Primarily, the detection of G. lamblia was associated with a growth restriction in certain three-month periods. Dietary fortification with LNS or corn-soy flour was not associated with the prevalence of the studied viruses or parasites among the children.
The main possible causes of bias in our study design were the symptomatic infections and the contamination of the samples during the laboratory analyses. For this reason, the guardians recorded on a daily basis the presence or absence of various illness symptoms. To detect possible contamination during the laboratory analyses, we used negative control samples in sample preparation, extraction and PCR but we found none. Also, the possibility of false negative results was reduced by the use of the positive control samples.
The strength of the trial was the high internal validity. This was due to the random group allocation, blinding of the outcome assessors and similarity of the intervention groups at enrolment. The success of faecal sample collection was relatively low (47.4%) which can be considered as a limiting factor in the study. However, this should not have biased our conclusions as the proportions with available sample were similar in all the intervention groups, also at 12 months of age when we obtained the lowest sample collection rate. Accordingly, we conclude that the sample findings are valid and representative of the target population from which the sample was drawn.
Of the studied microbes in our study, the presence of G. lamblia was mainly negatively associated with subsequent linear growth among asymptomatic infants and  young children. Such an association between G. lamblia and child growth has earlier been identified in cohort studies in Sub-Saharan Africa, Southern Asia and South America (17). Although there have also been studies in which this association was not found (18,19), the bulk of the evidence seems to suggest a link between symptomatic or asymptomatic Giardia infection and reduced linear growth in most lowincome contexts. Such an association is likely to be causal, as Giardia infections are often chronic (20) and can lead to prolonged aberrations in gut function and nutrient absorption (21,22). Additionally, systemic inflammation caused by chronic infections can interfere the growth hormoneinsulin-like growth factor 1 pathway and reduce growth plate activity and hence linear growth (21,23). In our sample, asymptomatic Cryptosporidium spp. carriage was associated with reduced linear growth only at 18 months of age. A recent meta-analysis of data from six studies suggested a linear growth restriction associated both with symptomatic or asymptomatic Cryptosporidium infection (24). Short episode duration may be the reason why in most cases, there was no association between the documented viral infections and children's length gain in the subsequent three-month period. The excretion of enterovirus, parechovirus, rotavirus, norovirus and rhinovirus typically lasts only some days to weeks. Furthermore, viral infections often elicit an interferon-based host response that provides short-acting protection also towards other viral species (25). A single time point assessment of a child's viral infection status is thus unlikely to be representative of her situation over the whole subsequent period of three months. If this was the case, the use of three-month follow-up could obscure the importance of viral infections in linear growth restriction. Few studies have addressed the association between asymptomatic viral infections and growth (26,27). To better assess the growth impact of non-chronic viral infections, one should thus probably use with a short follow-up period a more sensitive outcome variable, such as knemometry, plasma concentrations of insulin-like growth factor or collagen X (21,28,29). The finding of no association between the dietary supplement given to the children and detection of selected microbes in the children's stool is not surprising, since diet has not been reported to significantly influence viral propagation in children who are not malnourished. The result is also in line with an earlier intervention study, in which provision of LNS to children with severe acute malnutrition was not associated with a change in the detected virome in the children's stools (30). In a previously published research on the same study sample, the dietary intervention was not either associated with the composition of the children's microbiota (10).
Taken together, our results suggest that asymptomatic G. lamblia infections may restrict child growth in low-income settings such as rural Malawi. As a conclusion, asymptomatic viral infections are common and may also influence growth, but more refined measurement of growth or growth-related activity is necessary to better elucidate this question.