Microsporidia are predominantly intracellular single-celled fungi that lead intracellular lifestyles (1). Once classified as protists, microsporidia comprise about 220 genera and 1,700 species and possess highly reduced genomes, lack mitochondria, and have a polar tube that facilitates the infection of host cells; this infection creates a highly specific and obligate relationship with their eukaryotic hosts (2). Almost all animals, both vertebrates and invertebrates, can be infected by microsporidia. As opportunistic microorganisms, microsporidia thrive in immunocompromised individuals; however, an increasing number of species are recognized as emerging pathogens that persist in immunocompetent individuals (3, 4).

Currently, nine genera of microsporidia, Anncaliia, Encephalitozoon, Enterocytozoon, Microsporidium, Pleistophora, Nosema, Trachipleistophora, Tubulinosema, and Vittaforma, are known to infect humans. Among these, Enterocytozoon and Encephalitozoon are the most frequently reported in clinical settings (2, 3). They are considered opportunistic pathogens associated with diarrhea, malabsorption, and other systemic diseases and, historically, in persons suffering from AIDS (5). Disseminated microsporidia infection can be associated with high mortality in primates (6). Microsporidia generally follow single, host-specific life cycles except for a few, which can live in multiple hosts (2).

Microsporidia spores can be found in nearly all environments, persisting in surface and municipal water supplies (2). And because the spores are abundant and can be horizontally and vertically transferred, the aim of the current study was to identify and characterize their prevalence in the early human life. This understanding may help identify risks of infection and provide early therapeutic and/or interventional opportunities. This study utilizes a metagenomic sequencing approach to investigate the presence and persistence of microsporidia among cohorts of preterm infants and a publicly available term infant cohort. Importantly, this study is the first to report mitosporidia colonizing humans, particularly, premature and VLBW infants.

The study comprised of three cohorts, which are described in Table 1. Briefly, two in-house cohorts (1 and 2), comprising preterm and very low birth weight (VLBW) infants, were utilized, while the third cohort comprised 19 term infant samples, in duplicate, from a previously published dataset (7).

In comparing preterm and term infant cohorts, mitosporidium genus was found to be more abundant in the preterm cohorts (Supplementary Figure S1). PCA analyses revealed no significant clustering based on the batches, indicating limited batch effects and justifying the combined analyses of all samples (Supplementary Figure S2). No significant differences were noted in the distribution of microsporidia species among the three cohorts while comparing the preterm infants against the terms (Supplementary Figure S3). However, the distribution of microsporidia did differ between early life (week stage) and at two years of age. The PCA plot (Supplementary Figure S4) reveals a distinct distribution of microsporidia among children at 2 years of age compared to the preterm samples or compared to infants born at term (cohort 3). Upon comparing the microsporidia of preterm infants and at two years for the same samples from cohort 2, a few observations remain. First, some of the microsporidia were significantly increased (Figure 1) at 2 years of age than at the preterm infant stage (6 weeks). And second, in comparing the preterm and term cohorts, several microsporidia were found to be significantly different between the groups (Figure 2).

This study is the first, to our knowledge, to report the presence and distribution of various species of microsporidia during early human development. Cohorts one and two were collected from unrelated donors in the same neonatal intensive care unit (NICU) in the Tampa area, from two separate studies and 4–9 years apart. Cohort three includes samples from a previously published study conducted in an unrelated NICU, in a different city with different donors. Although it remains unclear whether the microsporidia are being transmitted horizontally or vertically, the distinct composition observed during prematurity—when infants are often underdeveloped and immunocompromised—deserves special attention. The dissemination of microsporidia in immunocompromised individuals can lead to severe consequences, such as malnourishment and growth restrictions, which can be fatal (see below). Infants born prematurely and sampled after 2 years seem to retain a unique microsporidia composition compared to infants born at term.

As obligate intracellular parasites, most microsporidia have reduced their genomes, and lost their mitochondria, instead depending on the host for ATP and energy production. Microsporidia are the only known eukaryotes to have lost nearly all the genes encoding the mechanistic target of rapamycin, or mTOR, which regulates cell division, energy harvest, and replication; the intracellular parasites instead deprive mTOR pathway components of its host cells (15). Thus, increased parasitic burden can lead to unprecedented risks for epithelial cell renewal and metabolic dysfunction in the intestines (1, 16, 17).

The spores of these parasites are uniquely characterized by having a polar tube or filament that is used to infiltrate host cells (1). While historically known to infect mostly arthropods and fish, these intracellular fungi have gained increased attention because of their additional ability to infect the intestinal epithelium of humans, thriving via dissemination in those that are immunocompromised, and recently recognized to be capable of broad-host range preferences (2, 16). Their infectious burden was originally described in one of the largest case studies among HIV patients (18–21) and remains a significant pathogen in most reported cases (2, 4, 22).

While vertical transmission has been observed in various animals, it has not yet been documented in humans (2). Yet, the ability to thrive in the food chain suggests that in addition to water, horizontal transfer of microsporidia is likely to occur in the food supply of humans (22). Of relevance to both immunocompetent and immunocompromised individuals, host immune factors regulating microsporidia infection have been shown to impact the susceptibility to bacterial pathogens in a non-human model system (23). Additionally, neurologic syndromes and microsporidiosis have been correlated following solid organ transplantation (24). Thus, it has been recommended that clinicians consider microsporidia burden (or infections) when encephalitis is suspected, even without gastrointestinal symptoms (2, 22, 24).

Microsporidia are known to cause significant diarrhea, wasting, and malabsorption (5), and among the immunocompromised, dissemination can be fatal (25). Increased burden, dissemination, and added complications associated with this intracellular pathogen should be considered in this vulnerable population of very and extremely low birth weight infants. With an emerging number of infections being reported in immunocompetent individuals (25, 26), compositional changes in microsporidia abundance should be considered in term infants that develop gastrointestinal symptoms that do not resolve. An important limitation of the current study is that 2-year samples were available only for VLBW infants and not for the term infant cohort. In addition, the sample size of our cohort is too low to reliably conclude on the abundances of microsporidia in these infant populations. Although the presence of microsporidia is generally not considered common in humans, this is not the first or only study to identify their presence at an early age. A previous study (27) reported the presence of microsporidia in preschool children. Based on this current study, it is difficult to ascertain if the microsporidia in premature infants were horizontally acquired from the NICU environment or vertically transferred during gestation or birth; however, to the best of our knowledge, this is the first report of persistent microsporidia identified in premature infants of disparate NICU environments. Follow-up investigations should examine if term infants retain certain microsporidia upon reaching two years of age, or if this finding is restricted to infants born VLBW.