Intestinal parasites can be broadly classified into protozoa (single cell organisms) and helminths (multicellular, known as worms).³⁹ Globally, there are an estimated 895 million people infected with soil transmitted helminths (STH). Intestinal protozoa (IP) have a lower overall prevalence rate, but still, over 350 million people are believed to be infected with 3 of the most common protozoan parasites40. Protozoan infections are common in low and middle income countries (LMICs). Food-chain globalisation, international travel and migration are leading to an increase in protozoan infections in high income countries, where they are more common than intestinal helminth infections.³⁹ 

DIARRHEAS CAUSED BY PROTOZOAN PARASITES

The most common intestinal protozoan parasites are Giardia intestinalis (Giardia duodenalis or Giardia lamblia), Entamoeba histolytica, Cyclospora cayetanensis, and Cryptosporidium spp. Diarrheal diseases caused by these pathogens are known respectively as giardiasis, amebiasis, cyclosporiasis and cryptosporidiosis.⁴¹​​​​​​​

DIARRHEAS CAUSED BY SOIL-TRANSMITTED HELMINTHS

Globally, the principal soil-transmitted helminths are the roundworm (Ascaris lumbricoides), the whipworm (Trichuris trichiura) and hookworms (Necator americanus and Ancylostoma duodenale). Symptoms experienced following helminth infection are related to the number of worms harboured: people with infections of light intensity (few worms) do not usually experience discomfort, whereas heavier infections can cause a range of symptoms including some that manifest in the intestine (diarrhea and abdominal pain), malnutrition, general malaise and weakness, and impaired growth and physical development. Soiltransmitted helminths contribute to the burden of diseases by impairing the nutritional status of the people they infect in a variety of ways: they feed on host tissues, cause intestinal blood loss and hamper the absorption of nutrients.⁴²

• Ascaris lumbricoides is the most common intestinal nematode that infects humans, with an estimated 807 - 1,221 million people infected each year. ⁴³ Infection commonly occurs without symptoms. The symptomatic form is characterized by an early lung phase followed by a later intestinal phase, which is characterized by diarrhea, mild abdominal pain, anorexia, nausea and vomiting.⁴¹
   
• An estimated 604-795 million people in the world are infected with Trichuris trichiura. People with heavy infections can experience frequent painful bowel movements that contain a mixture of mucus, water and blood.⁴⁴
   
• An estimated 576-740 million people in the world are infected with hookworms, usually without symptoms. Few people, especially those infected for the first time, experience gastrointestinal symptoms. The most common and serious effects of hookworm infection are intestinal blood loss leading to anaemia, in addition to protein loss.⁴⁵

MICROBIOTA: A ROLE IN THE MARKED CLINICAL VARIABILITY OF PARASITIC DIARRHEA?

Parasitic protozoan infections are characterized by marked variability in their clinical presentation: they can be asymptomatic or cause diarrhea, abdominal pain, weight loss, etc. Recent studies have highlighted the potential contribution of the intestinal microbiota to this clinical variation: for instance, an abundance of Prevotella copri in the gut microbiota predicted diarrhea in the context of Entamoeba histolytica infection;⁴⁶ low Megasphaera abundance prior to and at the time of Cryptosporidium detection was associated with parasitic diarrhea in infants in Bangladesh, suggesting that the gut microbiota may play a role in determining the severity of a Cryptosporidium infection.⁴⁷ In turn, infection by protozoan parasites alters the gut microbiome.^48,49

Regarding helminths, the complex interactions between worms and the microbiota (“two of humans’ old friends” ⁵⁰) are currently under study⁵⁰ (Figure7). Authors agree on the existence of a complex and dynamic interplay between parasite(s), the host microbiota and host immunity, capable of shaping the clinical outcomes of parasitic infections. ^46,48

Rotaviruses, norovirus, sapovirus, astrovirus, and adenovirus: five virus types are currently recognized as the main causes of viral diarrhea.²¹

Of the 2 billion plus episodes of diarrheal disease that occur across the world each year, as estimated by the 2016 Global Burden of Disease (GBD) Study,2 almost 900 millions of the moderate-to-severe episodes were attributed to just three of these viruses: rotavirus, norovirus and adenovirus²².

ROTAVIRUS, THE NUMBER ONE DIARRHEAL KILLER IN CHILDREN

Despite the development and availability of rotavirus vaccines since 2006,²² this virus, which causes more severe symptoms than most other enteric pathogens,²² was still responsible for over 228,000 deaths worldwide in 2016, of which over 128,000 occurred in children below the age of 5² – making rotavirus the leading cause of diarrhearelated mortalities among this segment of the population (Figure 4).

WATERY DIARRHEA

Whatever the virus that triggers an episode of diarrhea, the process of infection is broadly the same: the virus infects the epithelial cells of the small intestine and causes damage that hampers the absorption of fluid.²¹ Viral diarrhea usually manifests itself in the form of a watery (non-bloody) diarrhea. It can be accompanied by other symptoms, i.e. nausea, abdominal cramps, vomiting and fever,²² resulting in what is known as viral gastroenteritis.

REHYDRATION… AND PROBIOTICS

Just as for the other etiologies (bacterial or parasitic) of infectious diarrhea, management of viral diarrhea relies on oral or intravenous fluid replacement therapy, depending on the degree of dehydration.²¹ In addition, according to the latest conclusions of the ESPGHAN committee (2023),²⁰ healthcare professionals may recommend some probiotic strains (L. rhamnosus, S. boulardii and L. reuteri) for the management of acute gastroenteritis in children, since there is some evidence (certainty of evidence: low; grade of recommendation: weak) of reduced duration of diarrhea, and/or length of hospitalization, and/or and stool output.

IMPROVING ROTAVIRUS VACCINE EFFICACY, A CHALLENGE YET TO OVERCOME

Regarding prevention, the usual preventive measures apply (ensuring safe drinking water, adequate sanitation and frequent handwashing, limiting contact with infected people, etc.). Given the considerable burden of rotavirus diarrheal disease, rotavirus vaccines are another important preventive measure. ^22,23

These were estimated to have prevented 139,000 deaths from rotavirus among under-fives during the period 2006 to 2019, and to have prevented 15% of under-five rotavirus deaths in 2019.²⁴ However, vaccine efficacy is region-specific and displays poor seroconversion in low-and middle-income countries. Human clinical trial data have suggested a possible link between the gut microbiota and the enteric immune system’s response to rotavirus vaccine²⁵ (Figure 5).

MICROBIOTA: FRIEND OR FOE IN THE ONSET OF VIRAL DIARRHEA?

In cases of viral diarrhea, as with infectious diarrhea in general, the outcome of the confrontation between pathogen and host depends on complex balances that in large part involve the microbiota.

The gut microbiota displays bidirectional interactions with rotavirus and norovirus infections:¹⁴ it can either protect against or predispose the host to infection; in turn, an infection can alter the gut microbiota. Some bacteria seem able to inhibit viral infection. For example, one study shows segmented filamentous bacteria preventing and curing rotavirus infection in mouse colonies³⁵ (Figure 6). On the other hand, in vitro and in vivo studies indicate the gut microbiota’s involvement in facilitating viral infection: certain gut microbes (e.g. Enterobacter cloacae) stimulate the ability of human norovirus to infect human B cells in vitro; microbiota elimination by antibiotics delays infection, reduces infectivity and/or viral titer of norovirus and rotavirus in mice.^8,36

Therefore, any invasive pathogens might have different effects depending on the state of the gut microbiota. ³ The optimal microbiota profile and microbiota-targeting strategies that would lower the risk of infection and the following viral diarrhea remain to be characterized.³⁷

As to the effect of viral infection on gut microbiota composition, numerous studies have documented specific patterns of dysbiosis in patients suffering from viral diarrhea compared to healthy controls^25,38. A reduction of microbiota (alpha) diversity is often reported, but specific taxa increases or decreases vary widely across the studies.¹⁴ And one question remains: does the dysbiosis observed during viral diarrhea reflect a prior disposition that might have facilitated the infection, is it a state caused by the virus, or is it a combination of both?

Antibiotics are a powerful tool in the fight against bacterial infections, however they also disrupt the protective intestinal microbiota and this can lead to unintended consequences including antibiotic-associated diarrhea (AAD) in as much as 35% of patients.^17,18,19 The incidence of AAD depends on several factors: ^17,18,19 age (among children this percentage can reach up to 80%) ¹⁵, setting, type of antibiotic, etc. Most of the time, AAD is caused by antibiotic-induced dysbiosis, is of mild intensity and is self-limiting, lasting between 1 and 5 days.

While the etiologies for AAD are diverse, approximately one-third of AAD cases are attributed to C. difficile. Under certain conditions, C. difficile will trigger an inflammatory response leading to a range of clinical outlooks, from mild diarrhea to pseudomembranous colitis, toxic megacolon and/or death.¹⁷

Underlying how lethal bacterial diarrhea can be, these 8 bacteria were responsible for more than a third of the over 1.65 million deaths from infectious diarrhea recorded worldwide in 2016.²

FROM INFECTION TO DIARRHEA

The mechanisms that lead to bacterial diarrhea depend on the bacteria involved. Transmitted via contaminated food, water or by personto-person contact, Shigella infests the gastrointestinal tract, produces an enterotoxin and serotype toxin 1, destroying the intestinal epithelium and leading to severe bloody and mucous diarrhea.^3,5
Pathogenic variants of Vibrio cholerae produce a cholera toxin that activates anion secretion, inhibits absorption of electroneutral NaCl and destroys the intestinal barrier function, thereby inducing massive fluid secretion in the lumen of the small intestine and the loss of large amounts of water, sodium, chloride, bicarbonate and potassium.3,5,13 Different pathogenic E. coli strains, classified into different pathotypes (Table 1), cause mild to severe diarrhea usually accompanied by fever. E. coli adheres to the intestinal epithelial cells through the adherent fimbriae, produces toxins and exerts its pathogenic effects.^3,5

EFFECT OF PATHOGENS AND DIARRHEA ON MICROBIOTA

Bacterial diarrheas are accompanied by dysbiosis, usually with an overabundance of facultative anaerobes (Escherichia, Streptococcus, Enterococcus, etc.) in dysenteric diarrhea, and a depletion in bacteria of known immuno-modulatory effects (Lactobacillus ruminis, Bifidobacterium pseudocatenulatum)⁷ (Figure 3).

For example, in cholera, the gut microbiota is substantially modified both during and after infection, this as a consequence of the removal of the mucus layer along with the residing gut microbial community and of the delivery of toxin by V. cholerae. ¹³ During recovery, the gut microbiota of cholera patients slowly repopulates via an accumulation pattern similar to that of the maturation of the gut microbiota observed in children.³
In the same way, children infected with diarrheagenic E. coli (DEC) display a distinctive gut microbial composition, with a high fraction of Bacteroidetes and Proteobacteria and a reduced abundance of Firmicutes. ¹³ Increases in Proteobacteria could be partially explained by an increase in Escherichia/ Shigella species (as the cause of diarrhea) and in other members of Enterobacteriaceae, such as Citrobacter and Enterobacter (related to histamine production induced by proinflammatory environments and associated with E. coli adherence).¹⁴ Frequent antimicrobial use can also partly explain the observed dysbiosis.⁷

GUT MICROBIOTA PROTECTION AGAINST INFECTIONS

Symmetrically, the gut microbiota has demonstrated effects on bacterial infection. In germ-free animals, their lack of a gut microbiota and the absence of ecological competition results in an immature immune system that renders them highly susceptible to diarrheal pathogens: 10 colony-forming units (CFU) of Salmonella are enough to cause a lethal infection, whereas to kill 50% of mice with an intact gut microbiota 10³ –10⁹ CFU are required.⁸

In humans, Prevotella, Bifidobacterium and Blautia have been shown to reduce colonization of V. cholerae. Conversely, Paracoccus is believed to support the growth of the pathogen.¹³ This is the reason why promoting a ‘healthy’ gut microbiome has been considered a useful approach in the intervention and prevention of cholera.¹³

BACTERIAL AND YEAST PROBIOTICS, PREBIOTICS AND FMT

The severity of several bacterial infections could be reduced by probiotics: for example, probiotic E. coli inhibits the biofilm formation of other E. coli strains and likewise those of the pathogenic Staphylococcus aureus and S. epidermidis. ³ Regarding dysentery, a combination of Lactobacillus and Bifidobacterium strains and a Streptococcus strain reduces both the duration of blood-affected diarrhea and the time spent in hospital.³
Numerous mechanisms could explain why probiotics alleviate diarrhea:³ production of antimicrobial substances, competitive exclusion, competition with cell binding sites, production of acids and metabolites that lower the surrounding pH, strengthening of the gut mucosal barrier, modulation of gut mucosal immunity and gut microbiota diversity. For example, the probiotic yeast Saccharomyces boulardii may facilitate gut microbiota restoration in children with acute diarrhea.¹⁵

Prebiotics can also have a positive impact on diarrhea: by increasing the bacterial production of short-chain fatty acids (SCFAs) such as butyrate, which contribute to the gut barrier integrity; and by antagonizing the adherence of pathogens to epithelial cells, thus inhibiting colonization and promoting gut pathogen elimination.³

Fecal microbiota transplantation (FMT), which aims to restore a healthy gut microbiota, has proven effective and is only indicated in the treatment of recurrent C. difficile in both adults and children.¹⁴

INFECTIOUS DIARRHEA, ONE OF THE LEADING CAUSES OF INFANT MORTALITY WORLDWIDE

The passage of 3 or more loose or liquid stools per day corresponds to the common definition of diarrhea as set out by the WHO.1 Both criteria (frequency and consistency) are necessary: frequent passing of formed stools is not diarrhea, nor is the passing of loose stools by breastfed babies (Figure 1). 1.6 million deaths were attributed to diarrhea in 2016.² Children are particularly at risk: diarrheal disease is the 3rd leading cause of death in children under 5 years of age. A large part of the mortality used to be attributed to severe dehydration linked to fluid loss but today, septic bacterial infections account for an increasing proportion of all diarrhea-associated deaths.¹ Malnourished or immunity-impaired children are the most at risk of life-threatening diarrhea, as well as people living with HIV.¹

PATHOPHYSIOLOGICAL SYNDROMES OF DIARRHEA

From the clinical perspective, diarrheagenic pathogens can cause 2 pathophysiological syndromes:⁴

• Noninflammatory diarrhea (NID): patients present with nausea, vomiting, watery and voluminous stools, and abdominal cramping resulting from intestinal secretion (the intestinal mucosa remains intact). This milder course of disease is usually viral (Rotavirus, Norovirus…) but it can also be bacterial (enterotoxigenic Escherichia coli, Clostridium perfringens…) or parasitic (Giardia, …);
• Inflammatory diarrhea (ID): patients present with fever, abdominal pain, tenesmus and bloody stools of smaller volume than in NID. This severe course of disease is usually caused by invasive or toxinproducing bacterial strains (Shigella, Salmonella species…) that lead to mucosal barrier disruption and tissue destruction.

BEHIND THE SCENES OF DIARRHEA: THE MICROBIOTA

VICIOUS CIRCLE: WHEN DIARRHEA LEADS TO GUT DYSBIOSIS

Infectious diarrhea is regarded as a major dysbiotic event resulting from:

• increased bowel movements and disruption to mucosal integrity, ³
• increased proportion of water in the fecal matter and reduced transit time which contributes to taxonomic scarcity, ³
• and possible oral rehydration, zinc supplements, probiotics and even antimicrobials (in the case of dysentery or bacterial infections) which also contribute to an imbalance in the intestinal microbiota.⁷

Depending of the type of infection, infectious diarrheas are usually accompanied by dysbiotic states: ⁷ bacteria-induced diarrhea is generally linked to an elevation in Escherichia, Streptococcus and oral bacteria; viral infections lead to a less pronounced reduction in anaerobic commensals in the gut (higher abundance in Bifidobacterium); giardia-induced diarrhea is linked to a decrease in Gammaproteobacteria and an enrichment in Prevotella.

VIRTUOUS CIRCLE: WHEN THE GUT MICROBIOTA OFFERS PROTECTION

Mechanisms by which the gut microbiota provides colonization resistance can be both direct and indirect. The microbiota directly inhibits diarrheal pathogens mainly via competition for nutrients, but also by limiting in a variety of ways the growth of diarrheal pathogens: secreting bacteriocins (antimicrobial peptides), cell contact-dependent inhibitory structures (type VI secretion system), producing molecules that reduce the pathogens’ virulence…

The microbiota also indirectly inhibits diarrheal pathogens via its effects on the host: by promoting gut barrier maintenance and by stimulating both the innate and the adaptive immune system.⁸

HOW TO MANAGE INFECTIOUS DIARRHEA?

The majority of intestinal infections are self-limiting in immunocompetent individuals. Nevertheless, some patients (with severe dehydration, more severe illness, persistent fever, bloody stools, immunosuppression…) require specific diagnostic investigation. These can include a complete blood count, a creatinine and electrolytes assessment, verification of leukocytes and lactoferrin presence in the stools, stool culture, along with C. difficile testing, PCR, ova and parasites search, endoscopy and abdominal imaging¹¹. The American College of Gastroenterology (ACG) guideline¹² provides recommendations for the diagnosis and management of adult patients presenting with acute diarrhea of suspect infectious etiology (Figure 2). Clinical investigation in children is based on the same principles.²³ In 2023, World Gastroenterology Organisation Global Guidelines included probiotics in the prevention and treatment of some infectious diarrhea⁵⁷.

New study has identified a specific clade of the bacterium Fusobacterium nucleatum (sidenote: Fusobacterium nucleatum Fusobacterium nucleatum is a Gram-negative, anaerobic bacterium commonly found in the human oral cavity. It plays a role in periodontal diseases and has been associated with various infections and inflammatory conditions. Fusobacterium nucleatum is notable for its ability to adhere to and invade host tissues, facilitating interactions with other microorganisms and host cells. ) (Fn) that dominates the colorectal cancer (sidenote: Colorectal cancer Colorectal cancer is a type of cancer that begins in the colon (large intestine) or the rectum. It typically starts as small, benign clumps of cells called polyps that form on the inner lining of the colon or rectum. Over time, some of these polyps can become cancerous. ) (CRC) niche, revealing significant insights into the bacterium's role in cancer progression. ¹ This groundbreaking study, led by Fred Hutchinson Cancer Center researchers, uncovers genetic and functional distinctions in Fn strains associated with CRC, highlighting potential targets for therapeutic interventions.

Identifying the culprit

The research team collected and sequenced genomes from 135 Fn strains, including 80 from healthy oral cavities and 55 from CRC tumors. Using advanced genomic techniques, they performed a pangenomic analysis, which identified 483 genetic factors enriched in CRC-associated strains. The findings revealed that the Fn subspecies animalis (Fna), previously considered a single subspecies, is actually composed of two distinct clades: Fna C1 and Fna C2. Among these, only Fna C2 was found to dominate the CRC tumor niche, suggesting a unique role in cancer development.

Genetic and functional Insights

Further analysis showed that Fna C2 possesses 195 genetic factors associated with increased metabolic potential and gastrointestinal colonization, distinguishing it from Fna C1. This clade-specific genetic repertoire includes genes that enhance the bacterium's ability to invade host tissues and evade immune responses. The study also demonstrated that Fna C2-treated mice developed significantly more intestinal adenomas compared to those treated with Fna C1 or control groups. This suggests that Fna C2 not only colonizes CRC tumors but also actively promotes tumor growth.

Implications for treatment

These findings have profound implications for understanding the microbiome's role in CRC and developing targeted treatments. The study highlights the potential of focusing on Fna C2 for therapeutic interventions. For instance, identifying and targeting the specific metabolic pathways and virulence factors unique to Fna C2 could lead to new strategies for preventing or treating CRC. Additionally, the research underscores the importance of considering bacterial clades rather than species alone in microbiome studies, as significant differences can exist within subspecies.

Is there an axis of communication between the gut and germ cells? Yes, according to a new study published in Nature. ¹

Dysbiosis, harmful to the reproductive system and future children

Researchers at the European Molecular Biology Laboratory in Heidelberg and Rome induced gut dysbiosis in male mice by administering either non-absorbable antibiotics (which do not cross the gastrointestinal epithelium) or osmotic laxatives. They then mated them with healthy females and analyzed their offspring. 

Result: the pups suffered not only from lower birth weight and severe growth restriction, but also an early mortality rate 3.5 times higher than that of litters from healthy males.

Leptin, a key signaling factor?

Even more interestingly, in male mice mutant for the leptin gene (mimicking the effect of dysbiosis on leptin gene activity), the researchers noted characteristics similar to dysbiotic males (lower testicular weight and abnormal seminiferous tubules). Their sperm also showed changes in the abundance of certain "microRNAs" known to play a role in placental development during gestation.

By crossing these mutants with normal females, the researchers noted that the embryos showed a significant number of genes differentially expressed compared with embryos from healthy males. For the researchers, this demonstrates that leptin may well be an important signaling molecule in the gut-germline axis.
 

Severely disrupted placental development

Analysis of fetuses from mating with antibiotic-treated males and their placentas showed down-regulation of several important factors in placental development.

Among the main genes differentially expressed in placentas from crosses with antibiotic-treated males versus placentas from crosses with healthy males, the researchers found several clinical markers of placental insufficiency, such as those for pre-eclampsia.

Also present were:

• reduction of certain areas of the placenta (labyrinthine zone)
• altered placental vascularization
• a decrease in the placental growth factor

In a nutshell

For the first time, this study demonstrated that the environment can, via modulation of the gut microbiota, lead to sperm defects that can alter placental function and, therefore, the health of future children. It now remains to be seen whether a gut-germline axis also exists in humans... More to follow!

Should a man wishing to have children postpone conception if he has been prescribed antibiotics, which are likely to cause gut dysbiosis?

It is quite possible, according to a new study ¹ that, for once, highlights men's responsibility for the future health of their offspring. Conducted on mice, this study shows that males suffering from dysbiosis at the time of mating father unhealthy offspring. Fortunately, when the microbiota is rebalanced, this transmission no longer occurs.

Transgenerational effects of gut dysbiosis

To reach this discovery, researchers at the European Molecular Biology Laboratory ² in Heidelberg and Rome exposed male mice to 6 weeks of antibiotics. The aim was to induce dysbiosis (sidenote: Dysbiosis Generally defined as an alteration in the composition and function of the microbiota caused by a combination of environmental and individual-specific factors. Levy M, Kolodziejczyk AA, Thaiss CA, et al. Dysbiosis and the immune system. Nat Rev Immunol. 2017;17(4):219-232.   ) in the mice. They then mated them with healthy females, and the litters were analyzed after 3 weeks.

The result: compared to the offspring of fathers without dysbiosis (controls), mouse pups conceived by "dysbiotic" fathers:

• had a lower birth weight (a risk factor for diseases such as diabetes), 
• suffered more from stunted growth,
• had a higher mortality rate in the first 2 months.

To explain these surprising results, the scientists analyzed the reproductive systems of the antibiotic-treated males.

A direct impact on placental development

They noted that their testes had reduced size and sperm count and showed significant hormonal and metabolic changes. The sperm showed differences in certain molecules involved in the development of the placenta.

These data prove that the microbiota and germlines (the cells that transform into sperm) communicate with each other, and that there is therefore a "gut-sperm axis." 

• When analyzing the placentas of females mated with dysbiotic males during gestation, the researchers noted:
• poor placental vascularization, which could explain certain abnormalities in the offspring;
• the presence of markers similar to those of pre-eclampsia, a disease linked to abnormal placental development, responsible for intra-uterine growth restriction.

Towards a better pregnancy?

This is the first time that this kind of effect of the microbiota on the male reproductive system has been demonstrated in mammals. 

Is there also a gut-sperm axis in humans? As yet, this has not been confirmed, and further studies will have to be carried out. 

Considering that our lifestyles are likely to cause dysbiosis (poor diet, pollution, medications, etc.), proof of the existence of such an axis could pave the way for innovative approaches to ensure better reproductive health and more optimal pregnancies.