Site-Specific Incidence Rate of Blastocystis hominis and Its Association with Childhood Malnutrition: Findings from a Multi-Country Birth Cohort Study

ABSTRACT. In this study, we investigated the potential association between the burden of asymptomatic Blastocystis spp. (Blastocystis hominis) infection and nutritional status among children under 2 years of age using the data collected from 1,715 children from eight distinct geographic locations, including Bangladesh, Brazil, India, Peru, Tanzania, Pakistan, Nepal, and South Africa. Childhood stunting, wasting, and underweight were the outcome variables, and B. hominis infection was the exposure variable of this present study. The presence of B. hominis in nondiarrheal stools was evaluated by TaqMan Array Cards. Site-specific incidence rates were estimated using Poisson regression, and multiple generalized estimating equation was used to assess the association between the B. hominis infection and nutritional status. The site-specific incidence rates of asymptomatic B. hominis infections per 100 child-months were higher in Tanzania, Peru, and South Africa when compared with the other study sites. Moreover, in terms of site-specific association, childhood stunting was significantly associated with asymptomatic B. hominis infection in Bangladesh (odds ratio [OR]: 1.62; 95% CI: 1.26–2.08), India (OR: 1.78; 95% CI: 1.46–2.16), Nepal (OR: 2.26; 95% CI: 1.60–3.21), Peru (OR: 1.47; 95% CI: 1.26–1.71), South Africa (OR: 1.57; 95% CI: 1.35–1.83), and Tanzania (OR: 2.46; 95% CI: 2.18–2.79) sites. Wasting was associated with B. hominis in the Brazil site only (OR: 3.19; 95% CI: 1.31–7.77). On the other hand, underweight was associated in the Bangladesh (OR: 1.89; 95% CI: 1.48–2.42), Brazil (OR: 4.41; 95% CI: 1.57–12.4), Nepal (OR: 2.25; 95% CI: 1.52–3.35), and Tanzania (OR: 1.68; 95% CI: 1.42–1.99) sites. Our analysis further reveals that the presence of additional pathogens may play a pathogenic role in children who have B. hominis infection.


INTRODUCTION
Blastocystis spp. (Blastocystis hominis) is a widely distributed gastrointestinal protist that is commonly reported in tropical and subtropical countries. 1 This protist is one of the few intestinal parasites with a prevalence of more than 5% in the general population of developed countries and up to 30-60% in developing countries. 2 Based on epidemiological surveys, it is anticipated that this parasite has infected 1-2 billion individuals globally. 3 According to recent microbiome studies, this protozoan parasite may colonize the human gut for long periods of time without causing symptoms, and B. hominis carriers have more bacterial diversity than non-B. hominis carriers, implying that it should be regarded as a commensal rather than a pathogen. 3 Additionally, this organism has been associated with indicators of a healthy gut microbiota. 1 However, it has also been associated with a decrease in protective bacteria in the feces, such as Bifidobacterium spp. and Faecalibacterium prausnitzii. 1 Moreover, other researchers have attributed B. hominis to irritable bowel syndrome and inflammatory bowel disease as well as watery diarrhea, stomach pain, meteorism, a lack of appetite, and constipation. 2 Furthermore, B. hominis may have a pathogenic role in immunocompromised patients. 2,4,5 Studies have linked the parasite to cutaneous disorders and chronic or acute urticaria, in addition to atypical gastrointestinal symptoms. 2 The organism causes no symptoms in most people who carry the parasite, but it can also be found in people who have diarrhea or other digestive issues. 1 As a result, B. hominis can be an opportunistic organism that can have a negative impact on gut health when other pathogens are present. 1 Hence, evidence of its pathogenicity in humans is speculative and inconclusive. There is insufficient evidence to determine if B. hominis causes disease or is beneficial to gut microbial diversity. 1 Although there is still debate about its pathogenicity, recent research supports the pathogenicity of B. hominis in causing clinical symptoms in the absence of other intestinal infections. 6 Children with recurrent diarrhea had a higher prevalence of intestinal parasitic infection, implying that parasitic infection should be investigated in cases of chronic or recurrent diarrhea. 6 Moreover, intestinal parasite infections are most common in children of school age and may have a negative impact on growth. 7 Similarly, prior literature revealed that a higher cumulative burden of diarrhea prior to the age of 24 months was linked to a higher prevalence of stunting at that age. 8 In the absence of other pathogens, chronic diarrhea, nausea, anorexia, vomiting, dizziness, and flatulence caused by B. hominis have been found often in cases where the organism exists at higher numbers. 9 This research backs up previous findings that B. hominis can cause diarrhea in children in underdeveloped nations. 9 However, the precise role of B. hominis as a causative agent of growth faltering remains poorly understood. The findings of a study conducted among malnourished Bangladeshi adults imply that the presence of B. hominis in the human intestine has an impact on gut health and may play a pathogenic role in the presence of other pathogens. 1 Hence, there could be a possible association of B. hominis with all forms of malnutrition among children from resource-constrained settings.
Therefore, we have structured this study with the purpose of investigating the site-specific incidence rates of B. hominis and its possible association with all forms of malnutrition among children aged 1-24 months.

MATERIALS AND METHODS
Study design and ethical statement. The multi-country birth cohort Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health (MAL-ED) study, which was conducted across eight sites in South America, sub-Saharan Africa, and Asia (Bangladesh, Brazil, India, Nepal, Peru, Pakistan, South Africa, and Tanzania), has been previously described. 10 In summary, from November 2009 to February 2012, 1,715 children were registered from the community within 17 days of birth at all eight sites and observed for up to 24 months. Each of the eight study sites' institutional review boards gave approval to the study. 10 Every child's parents or legal guardians provided written, informed, and voluntary consent. All study protocols were carried out in accordance with the ethical requirements set by the regulatory authorities at each study location.
Collection of anthropometric, sociodemographic, and morbidity data. In accordance with 2006 WHO standards for children, anthropometric indices of length-for-age z-score (LAZ), weight-for-age z-score (WAZ), and weight-for-length z-score (WLZ) were derived using data from monthly anthropometric measurements taken up to the age of 24 months, using standard scales (secaGmbH & Co. KG, Hamburg, Germany). 11 Anthropometric data, as well as information on birth history, household demographics, and maternal factors, were obtained from study participants across all sites at the time of recruitment. 10 During household visits, which were conducted twice weekly, a full description of any morbidity and child feeding practices was acquired. 12 Starting at the age of 6 months, socioeconomic data were collected every 6 months. The WAMI (water, sanitation, hygiene, asset, maternal education, and income) index, which spans from 0 to 1, is a socioeconomic status index for households that was calculated thereafter. 13 A greater WAMI suggested a higher socioeconomic class. 14 The WHO criteria for better water and sanitation were followed, 15 and drinking water treatment was defined as filtering, boiling, or adding bleach. 16 Outcome variables. The outcome variables of the present study were stunting, wasting, and underweight. Stunting, also known as linear growth faltering, occurs when a child's LAZ falls below 22 SD, according to the WHO child growth standards (LAZ ,22). Weight-for-length z-score was less than 22 SD (WLZ ,22) for wasted children, and weight-forage z-score was less than 22 SD (WAZ ,22) for underweight children. A study participant's stunting, wasting, or underweight indicated an event of malnutrition.
Collection of stool samples and assessment of enteropathogens. Nondiarrheal stool samples were obtained on a monthly basis after enrollment and up to the age of 24 months at all study locations. In this investigation, no diarrheal samples were analyzed. Upon collection of these stool samples, no fixative was added to the stool samples, and the unprocessed stool aliquots were stored in 280 C freezers before being analyzed in the laboratory. 17,18 Using procedures described elsewhere, TaqMan array cards, a customized multiplex quantitative polymerase chain reaction using compartmentalized primer-probe assays, were used to detect a possible 29 pathogens from each of the samples. 19,20 As stated previously, the quantification cycle value of 35 was set as a threshold for the analysis. 19 In our study, we analyzed the occurrence of the 18S rRNA gene of B. hominis, and positive cases were checked for co-infection with other distinct pathogens, including Campylobacter jejuni, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), norovirus, atypical enteropathogenic E. coli (aEPEC), Shigella/enteroinvasive E. coli (EIEC), and Cryptosporidium spp. 21 Statistical analysis. Stata software (Release 14; Stata-Corp LP, College Station, TX) was used to analyze the data. Line graphs were used to illustrate the data in this study, including a line graph to assess the prevalence status of outcome variables with time and a variable for asymptomatic B. hominis infection ( Figure 1). We used frequency and proportion for qualitative variables and mean and standard deviation for quantitative symmetric variables to summarize the data. Poisson regression was used to calculate the incidence rate in all of the study sites. The association between asymptomatic B. hominis infection and three forms of malnutrition (stunting, wasting, and underweight) was studied using multiple generalized estimating equations (GEEs) with binomial family, logit link function, and exchangeable correlation. The WAMI (water/sanitation, assets, maternal education, and income) index, child's sex, maternal height, mothers' having fewer than three living children, and site were adjusted in the multiple GEE model. 22 Because of the birth cohort research, the child's age was used as a time variable in the model. Additionally, a P value of , 0.05 was used to portray significance, and the 95% CI was used to describe the findings.

RESULTS
General characteristics. A total of 1,715 participants were enrolled throughout the eight study sites, and 34,622 monthly nondiarrheal stool samples were collected from the participants over the course of the study, commencing with enrolment after birth. The demographic characteristics of the study participants (N 5 1,715) enrolled at each of the eight study locations are displayed in Table 1. Due to the unavailability of a subset of length measurements at the Pakistan site for this study, we eliminated children from that site (N 5 246) for our analysis.
Associations between B. hominis and other pathogenic enteropathogens. Table 3 Table 2 summarizes the strength of the co-infection associations between B. hominis and other pathogens.
Association between B. hominis infection and all forms of childhood malnutrition. Table 4 shows the results of a separate analysis involving the association between the burden of B. hominis infection with all forms of malnutrition (stunting, wasting, and underweight) during 1-24 months of age. After adjusting for the relevant covariates, the overall infection of asymptomatic B. hominis infection over the study period across all study sites, excluding Pakistan, was found to have a significant association with stunting (OR: 1.91; 95% CI: 1.79-2.05; P , 0.001) and underweight (OR: 1.41; 95% CI: 1.29-1.54; P , 0.001); however, no association was observed with wasting (OR: 0.94; 95% CI: 0.77-1.15; P 5 0.556).
When we looked at site-specific associations, stunting was found to be associated positively with the burden B. hominis infection over the study period across sites, including Bangladesh

DISCUSSION
According to epidemiological data, B. hominis is highly prevalent in tropical and subtropical settings, particularly in developing countries with inadequate hygiene conditions and consumption of contaminated food or water. Although its pathogenic potential is still questioned, B. hominis is commonly recognized as a pathogenic contributor to diarrhea, abdominal pain, and irritable bowel syndrome in people. In particular, in immunocompromised patients, such as those with HIV/AIDS or cancer, B. hominis infections can cause noticeable diarrhea. 23,24 In this current literature, the association between asymptomatic B. hominis infection and various forms of childhood malnutrition (i.e., stunting, wasting, and underweight) in children enrolled in the MAL-ED birth cohort study were analyzed. Our data demonstrated a considerable discrepancy in the prevalence of asymptomatic B. hominis infections when compared with the other study sites, with incidence rates per 100 child-months being higher in Tanzania, Peru, and South Africa (Figure 2). Further observations into our site-specific assessment revealed a significant association between asymptomatic B. hominis infection and childhood stunting in Bangladesh, India, Nepal, Peru, South Africa, and Tanzania. To the best of our knowledge, this is the first study to investigate the associations between the protist B. hominis and all forms of malnutrition in children from birth to 2 years of age.
When compared with the other study sites, our findings revealed a significant disparity in the prevalence of asymptomatic B. hominis infections, with incidence rates per 100 child-months being higher in Tanzania, Peru, and South Africa. These findings corroborate previous findings from epidemiological data that B. hominis is highly abundant in tropical and subtropical settings, particularly in developing countries with poor personal hygiene, inadequate hygiene conditions, and consumption of contaminated food or water. 1,23,24 According to our findings, the greatest incidence rate of B. hominis infection in children was reported in Tanzania (34%). The incidence of B. hominis found in this study is consistent with previous MAL-ED research on other enteropathogens, such as enterotoxigenic Bacteroides fragilis, enteropathogenic E. coli, and enteroaggregative E. coli, which found that the highest incidence rates were also found in Tanzania. [25][26][27] Another study revealed a seasonal effect on B. hominis prevalence, which rose to 23.2% in the summer, compared with 13.7% in the winter. 28 Summer waterbased recreational activities may also be involved because human fecal contamination has been linked to B. hominis loads in recreational rivers, implying a higher risk of parasite infection in the summer. 28 This seasonal pattern could explain B. hominis peaking in hot weather, and the prolonged summer season in Tanzania could be the underlying reason for the higher B. hominis occurrence in this particular study site.
Moreover, in our site-specific analysis, sites including Bangladesh, India, Nepal, Peru, South Africa, and Tanzania delineated a significant association between asymptomatic B. hominis infection and childhood stunting. According to a recent study, B. hominis infection can cause growth retardation, deterioration in cognitive and learning abilities, and a reduction in children's quality of life. 24 Infection with B. hominis is becoming increasingly widely recognized as a severe threat to human health, and some B. hominis subtypes are pathogenic. 24 Moreover, our result is consistent with a recent cross-sectional study of Australian aboriginal children aged 0-2 years that revealed an inverse relationship between B. hominis infection and height-for-age z-scores. 29 Only Brazil depicted a significant association between B. hominis infection and wasting. On the other hand, a significant sitespecific association between B. hominis and underweight was observed in Bangladesh, Brazil, Nepal, and Tanzania. Although there has not been a prior study tying the negative association between B. hominis infection and children's WLZ, it can be interpreted that, in the presence of other pathogens, B. hominis damages the intestinal mucosa, resulting in reduced nutrient absorption and growth failure. 1 A prior study conducted in Slovenia revealed the presence of B. hominis in fecal samples of diarrhea patients who had no  The findings also confirm that B. hominis meets all of the pathogenicity criteria set forth by Giardia spp. and E. histolytica, including the fulfillment of Koch's postulates. 32 Another study identified a link between the presence of amoebic forms and B. hominis protease activity. 33 The amoebic form of the protist B. hominis has previously been hypothesized to have a sticky surface that aids adherence to the intestinal epithelial cell lining. 33 This adhesion could make it easier for amoebic forms to release proteases that degrade external membrane, similar to what was demonstrated in E. histolytica. 33 Proteases have already been implicated as one of E. histolytica's pathogenic factors. 33 Entamoeba histolytica causes invasive intestinal disease that manifests as cramping, abdominal pain, and watery or bloody diarrhea over several weeks. 34 Consequently, E. histolytica-associated diarrheal illness was found to be negatively associated with preschool children's linear growth in a study conducted in an urban slum in Mirpur, Dhaka. 35 Therefore, it is probable that B. hominis is associated with diarrheal sickness and subsequent stunting in children.
Blastocystis hominis is disseminated via the fecal-oral pathway. 1 Earlier studies have shown that unsanitary environmental factors, as well as crowded houses in developing countries, contribute to enteric pathogen transmission. 1 Nonetheless, malnourished people have a higher chance of getting infected with enteropathogens. It is possible that a weakened immune system makes people more vulnerable to gastrointestinal infections. 1 According to our data, the risk of infection with B. hominis is 12% higher in the presence of C. jejuni. A prior study found a significant association between Campylobacter spp. and B. hominis. 29 This correlation revealed that the presence of Campylobacter spp. favors the presence of B. hominis and that the lack of one favors the absence of the other. 29 Our study also delineated that the odds of infection with B. hominis were higher in the presence of pathogenic enteropathogens such as Giardia spp., Cryptosporidium spp., ETEC, and Shigella/Shiga toxin-producing E. coli (STEC) by 62%, 47%, 31%, and 21%, respectively. In Beni-Suef Governorate, Egypt, a prior study discovered a significant prevalence of intestinal parasite infection, particularly Giardia-B. hominis co-infection, in diarrheic young children (up to 12 years). 36 This co-infection could be due to both parasites having the same environment, social conditions, transmission pattern, and age-prevalence curves. 36 In another study, G. intestinalis was also reported to be the parasite most frequently associated with B. hominis. 36 Tanzania. Adjusted in generalized estimating equation for sex; WAMI (water, sanitation, hygiene, asset, maternal education, and income) index, maternal height, mother having fewer than three living children and as co-pathogen infection, and site for overall estimates. Dependent variable: stunting (length-for-age z-score , 22 SD), wasting (weight-for-length z-score , 22 SD), and underweight (weight-for-age z-score , 22 SD). Independent variables: B. hominis infection. * Adjusted for sex; WAMI (water, sanitation, hygiene, asset, maternal education, and income) index, maternal height, and mother having fewer than three living children.
Furthermore, according to another study, largely asymptomatic Giardia spp. infections appeared to impair child growth, most likely due to malnutrition, while a similar link between Giardia spp. infection and nutritional status, as evaluated by anthropometric parameters in the Brazilian Amazon was recently discovered. 7 This co-infection could also explain why B. hominis may be associated with impaired linear growth among children. Co-detection of B. hominis with Cryptosporidium spp. has also been observed in previous studies. 37,38 Furthermore, Cryptosporidium spp. infection has been associated with stunting in numerous cross-sectional studies and a meta-analysis undertaken as part of the Global Burden of Disease Study. 39 Furthermore, prospective studies on three continents (Africa, Asia, and South America) have shown that Cryptosporidium spp. infection in infancy and early childhood is a cause of stunting. 39 We also observed an increase in the prevalence of B. hominis infection in the presence STEC, of another gut pathogen. 40 Our analysis also exhibited that the odds of B. hominis infection are higher in the presence of ETEC, aEPEC, and tEPEC (organisms known for causing gut infections and altered intestinal health). 26,41 The presence of the enteropathogenic E. coli (EPEC) genomic subtypes tEPEC and aEPEC was similarly inversely related to childhood linear growth in an earlier MAL-ED investigation. 26 Conversely, in the presence of norovirus and EAEC, the risk of B. hominis infection decreases. This finding backs up prior claims that B. hominis is an opportunistic organism. 1 Limitation. We have primarily evaluated the association between B. hominis and other pathogens in this study. The causality of this association, however, has not been investigated. Moreover, we have not measured sCD14 or any other marker of barrier dysfunction. Future research should thus concentrate on and take into account the aforementioned criteria to validate the association.

CONCLUSION
When compared with other sites, we found that the incidence rate of asymptomatic B. hominis infections was greater in Tanzania, Peru, and South Africa. Furthermore, FIGURE 2. Site-specific prevalence of stunting, wasting, and underweight by children's age in months. ZAMAN WAHID AND OTHERS our site-specific analyses in Bangladesh, India, Nepal, Peru, South Africa, and Tanzania found a significant link between B. hominis and childhood stunting. In conclusion, findings from the present study imply that B. hominis has a potential pathogenic role in gut health and negatively affects growth development. Moreover, the pathogen appears to have harmful functions in the presence of certain enteric pathogens well known for gut infection. As a result, we urge the association of this ambivalent pathogen with growth failure as well as other potentially harmful enteropathogens to be investigated further, with a focus on the identification of B. hominis subtypes.