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GI pathogen deep dive

Explore the biology, epidemiology and diagnostic options for common GI pathogens

We all know a wide range of bacteria, viruses and parasites can cause gastrointestinal illness. But are you familiar with the major E. coli pathotypes linked to human disease and how they differ? Do you know the challenges involved in identifying many GI pathogens in the lab, including Entamoeba histolytica and Campylobacter spp? Welcome to our deep dive into GI pathogens. Read on to learn about some of the common pathogens that cause gastrointestinal infections.

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Deep dive #1: E. coli pathotypes

Microbial, Microbe, Microbiome, Microbiota, Microbial DNA qPCR
E. coli are rod-shaped, gram-negative bacteria. While these bacteria form part of the normal gastrointestinal microbiome, several pathotypes can cause disease. These pathogenic varieties are responsible for hundreds of thousands of cases of gastrointestinal illness every year. While the signs and symptoms of these infections often overlap, the most effective means of clinically managing them can differ.

Four important E. coli pathotypes

Not all pathogenic E. coli strains are the same. Some cause more severe disease than others, which is determined by distinct genetically-encoded virulence factors. There are five important E. coli pathotypes implicated in human disease. These can be distinguished from one another using molecular assays that detect their unique virulence factor genes.

1) Enteropathogenic E. coli (EPEC)
2) Enterotoxigenic E. coli (ETEC)
3) Shigella/Enteroinvasive E. coli (EIEC)
4) Shiga-like toxin-producing E. coli (STEC), including O157:H7 serotype
1) EPEC

EPEC are classified into typical (tEPEC) and atypical (aEPEC) strains based on the presence of the adherence factor plasmid (pEAF). While both strains are important causes of diarrhea, tEPEC can cause more severe illness, leading to aggravation of malnutrition and persistent diarrhea. 

EPEC’s key virulence factor is the eae gene. eae encodes a protein that allows the bacteria to adhere to human epithelial cells within the GI tract, causing destruction of cells. 

In most cases, EPEC infections can be treated with hydration, although antibiotics are recommended for persistent infections or individuals at risk of severe infection (1).

EPEC’s key virulence factor is the eae gene.
2) ETEC

ETEC is the most common E. coli pathotype associated with diarrhea and is the leading cause of diarrhea among travelers and children. Its clinical symptoms range from mild diarrhea to a severe, cholera-like syndrome. 

The bacteria is characterized by the production of heat-labile (LT) and heat-stable (ST) enterotoxins. These toxins are released after colonization in the small intestine and lead to watery diarrhea (2). 

ETEC is usually self-limiting and can be treated with supportive care, including hydration. Antibiotic treatment can shorten the duration of diarrheal illness, but ETEC can be resistant to common antibiotics.

ETEC produce LT and ST enterotoxins, which cause symptoms.
3) Shigella/EIEC

EIEC is an invasive strain of E. coli that is very closely related in virulence and other properties to Shigella. Both are characterized by profuse, sometimes bloody diarrhea, high fever and stomach cramps. Shigella causes an estimated 450,000 infections in the USA each year, with an estimated $93 million in direct medical costs (3).

Shigella/EIEC causes illness by penetrating epithelial cells and destroying the integrity of the epithelial lining (1). 

While antibiotic treatment is recommended for severe shigellosis, drug resistant Shigella infections have been on the rise since 2016, with nearly 46% of infections being resistant in 2020 (4). Anti-diarrheal medications are not recommended, as these can worsen symptoms (1).

EIEC and Shigella cause illness by destroying the integrity of the epithelial lining.
4) STEC

An estimated 265,000 STEC infections occur each year in the USA (5). Symptoms of STEC range from mild intestinal disease to hemorrhagic diarrhea. This can ultimately progress to a potentially fatal condition known as hemolytic uremic syndrome (HUS), especially in the very young and old. Approximately 5–10% of patients with STEC infections develop HUS. 

The main virulence factors for STEC are the shiga toxins 1 (stx1) and 2 (stx2). STEC can carry either one or both toxin genes. 

There are more than 400 serotypes of STEC, of which O157:H7 is the most common. The O157 serotype has resulted in large-scale outbreaks in several countries and is commonly associated with HUS (6). Identifying O157 and non-O157 E. coli serotypes is important for infection control and epidemiological surveillance.

In some cases, STEC infection can progress to a potentially fatal condition known as hemolytic uremic syndrome (HUS).

Reducing the risk of HUS and severe GI disease

HUS is characterized by hemolytic anemia, thrombocytopenia and acute renal failure. Individuals at risk of developing HUS must be identified early, as their prognosis is dependent on the avoidance of antimicrobials, quick initiation of adequate supportive treatment and close monitoring.

Antimicrobial treatment is not recommended during STEC infection, as this can lead to an increased risk of HUS (5). This makes treating severe GI infections challenging, as the symptoms of STEC often overlap with other bacterial infections that are treated with antibiotics. 

A definitive diagnosis can mean the difference between effective and potentially harmful or ineffective therapy. Molecular assays that can distinguish between the main E. coli strains and specifically identify the presence of stx can help identify individuals at risk of severe GI infection. This can help reduce the risk for serious complications and improve overall patient outcomes.

Deep dive #2: Identifying challenging GI pathogens

Many of the difficult-to-culture or difficult-to-identify organisms listed below can be easily missed in the lab using standard diagnostic methods. This is because the unique culturing conditions and tests needed to diagnose them are not routinely performed. More sensitive and reliable molecular assays like multiplex PCR may provide a better diagnostic yield for these and other GI pathogens. In fact, both Nebraska Medicine and the Mayo Clinic recommend multiplex PCR panel tests for certain patient populations (7,8).

Now, take a tour through some of the most challenging GI pathogens to identify in the lab.

Four GI pathogens that are especially difficult to work with and identify in the lab

1) Campylobacter spp.
2) Shigella
3) Salmonella spp.
4) Intestinal parasites: Cyclospora cayetanensis,
Cryptosporidium, Entamoeba histolytica, Giardia lamblia
1) Campylobacter spp.

 

Campylobacter is estimated to cause 170 million cases of diarrhea each year (9). Symptoms of infection include bloody diarrhea, fever, abdominal cramping and vomiting.

Campylobacter adheres to intestinal epithelial cells in the GI tract, leading either to asymptomatic colonization or diarrheal illness (10).

Campylobacter is a fastidious organisms that grows most successfully under strict atmospheric conditions and at a temperature of around 42̊C (11). This can present a challenge for culture-based Campylobacter identification, as these are non-standard culturing conditions for bacteria.

An alternative method for identifying Campylobacter is the stool antigen assay. However, existing assays exhibit limited sensitivity and low positive predictive value (12).

Campylobacter is a fastidious organisms that grows most successfully under strict atmospheric conditions.

2) Shigella

Shigella is a gram-negative bacteria that causes over 165 million cases of illness worldwide each year.

While it is classically diagnosed using stool culture, this method has been shown to miss a large proportion of positive cases. Like Campylobacter, Shigella is a fastidious bacteria that is highly sensitive to changes in temperature and pH. A significant loss of pathogen viability occurs while samples are transferred from the clinic to the lab. In addition, Shigella culture is negatively impacted by patient antibiotic treatment prior to sample collection (13).

Shigella is highly sensitive to changes in temperature and pH.

3) Salmonella spp.

Salmonella are rod-shaped, non-spore forming gram-negative bacteria and can be divided into typhoidal and non-typhoidal serotypes (14).

There are 100 – 200 million cases of non-typhoidal salmonellosis each year (15). Non-typhoidal salmonellosis is characterized by watery diarrhea, fever, abdominal pain and vomiting.

The gold standard test for Salmonella infection is culture. However, this method is time- and labor-intensive, involving multiple 24-hour pre-enrichment steps and up to five days of total culturing time. In addition, culture exhibits low and variable sensitivity (16).

Salmonella culture requires multiplex 24-hour pre-enrichment steps – and up to five days of total culturing time.

4) Intestinal parasites: Cyclospora cayetanensis, Cryptosporidium, Entamoeba histolytica and Giardia lamblia

Microscopy remains a staple of the parasitology lab. Highly experienced laboratory technicians can identify parasites at different life-cycle stages based on their morphology and staining characteristics.

However, this technique is not without its challenges. For example, identifying Entamoeba histolytica using microscopy alone can be exceptionally difficult. As this parasite is only sporadically shed, multiple stool samples must be examined over a 10-day period.

Identification is also complicated by the similarity in morphology between E. histolytica, other related species and certain human cells. Under the microscope, E. histolytica cannot be differentiated from nonpathogenic species like E. dispar. In addition, certain E. histolytica life-cycle stages are difficult to distinguish from human macrophages and neutrophils.

It’s estimated that up to 40% of E. histolytica infections are missed using microscopy alone (17).

Similarly, Cyclospora and Cryptosporidium oocytes can be easily missed under the microscope due to their low shedding levels and/or difficult-to-identify morphology (18).

Lateral flow immunochromatographic assays can also be used to detect intestinal parasites. At the moment, however, there are no commercially available assays for Cyclospora.

It’s estimated that up to 40% of E. histolytica infections are missed using microscopy alone.

False-positive results

False positive results may be linked to sample contamination. They can have serious negative consequences for patients and healthcare institutions.

Patients may receive unnecessary or inappropriate changes in their treatment. For example, as discussed in deep dive #1, patients with a STEC infection should not be given antibiotics, as this can worsen their illness. As a result, a false-positive diagnosis could prove detrimental for these patients.

In addition, many GI pathogens are highly contagious and the presence of these illnesses can have ramifications for hospital function, including ward closure (19). False positives further run the risk of triggering public health alerts or investigations in the event that a pathogen may be foodborne or has human outbreak capacity.

False-negative results

False negative results can be caused by a low quantity of pathogen in the sample.

In many cases, individuals with GI illness will be placed on supportive or empiric treatment. However, if positive cases are not identified early, patients may not receive targeted treatment in a timely manner.

More to explore

References
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  2. Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2(2):123-140. doi:10.1038/nrmicro818
  3. Questions & Answers | Shigella – Shigellosis | CDC. https://www.cdc.gov/shigella/general-information.html
  4. CDC. COVID-19: U.S. Impact on Antimicrobial Resistance, Special Report 2022. Atlanta, GA: U.S. Department of Health and Human Services, CDC; 2022. https://www.cdc.gov/drugresistance/covid19.html
  5. Questions and Answers | E. coli | CDC. https://www.cdc.gov/ecoli/general/index.html
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