Breath Testing for SIBO: Limitations and Considerations

Published on


Diagnosis of small intestinal bacterial overgrowth (SIBO) has increased considerably in recent years, particularly due to its close association with irritable bowel syndrome (IBS). Yet the prevalence of SIBO is difficult to determine given methodological issues with testing. Read on to learn the various types of tests available and why breath testing remains the best option for clinical practice, despite its limitations.

While microbes are present throughout the gastrointestinal tract, a healthy small intestine contains relatively few microbes. Small intestinal bacterial overgrowth (SIBO) refers to an excessive concentration of bacteria in the small intestine. This condition has proven difficult to define and measure, particularly due to lack of a standardized test protocol and interpretation. Most studies have not evaluated bacterial load in healthy volunteers, and those that do typically find that 6 to 16 percent of healthy volunteers test positive for SIBO (1).

In this article, I’ll review the pathophysiology of SIBO, discuss the most common tests used in research and clinical laboratories, and share my recommendations for maximizing SIBO test accuracy.

The Pathophysiology of SIBO

A healthy colon, or large intestine, is an environment rich in microbes that aid in digestion, shape immune tolerance, and protect against pathogenic infection. In contrast, a healthy small intestine contains few microbes, allowing nutrients to come in more direct contact with nutrient transporters on epithelial cells. This low bacterial load is maintained by several mechanisms, including the acidity of chyme released from the stomach, antimicrobial peptides released by the small intestinal epithelium, and the migrating motor complex (2).

When these mechanisms break down, certain populations of bacteria can overgrow in the small intestine. Bacterial fermentation in the small intestine can produce a wide variety of complications. The most obvious are gastrointestinal symptoms such as bloating, chronic diarrhea, and abdominal distention. But SIBO can also lead to deficiencies of nutrients including vitamin B12, vitamin A, vitamin D, and vitamin E. Untreated SIBO can therefore lead to diseases of malnutrition like megaloblastic anemia, peripheral neuropathy, osteoporosis, and night blindness (3). SIBO is also associated with a number of other diseases, including diabetes, celiac disease, hypothyroidism, gastroparesis, chronic pancreatitis, and chronic kidney disease (4).

For the rest of this article, we’ll focus on testing for SIBO. For more on the pathophysiology of SIBO, check out this article published on my blog or sign up for my SIBO seminar.

How to improve the accuracy of SIBO breath tests.

Small-Bowel Aspiration and Quantitative Culture

There are two primary ways of testing for SIBO. The current “gold standard” test is small-bowel aspiration and quantitative culture. Aspiration is performed through an endoscopically or fluoroscopically confirmed guidewire-placed sterile catheter, and the sample must be promptly transferred to the appropriate laboratory for aerobic or anaerobic culturing. Considerable variability exists in methodology, and most studies lack validation against healthy controls.

The use of this test is largely limited to research settings because of its high cost, invasive nature, time commitment, potential for sample contamination, lack of standardization, and need for dedicated laboratories (5). SIBO is also patchy in its distribution throughout the small intestine (6), so a sample taken from one small section could return a negative result when a section directly adjacent is the one with bacterial overgrowth. Aspiration and quantitative culture also likely underestimates the bacteria present, since many species that live in the small intestine cannot be effectively cultured (7).

Though rarely used in clinical settings, this method remains widely utilized in research settings. Most experts have accepted a bacterial count of 105 cfu/mL aspirate or more to be diagnostic of SIBO (7, 8).

Breath Testing for SIBO

In contrast to quantitative culture, breath testing provides a simple, noninvasive, and widely available testing modality for suspected SIBO. Breath testing is based on the idea that bacterial fermentation of non-absorbed carbohydrates is the sole source of hydrogen and methane in exhaled breath (9, 10). Measurement of methane in addition to hydrogen can increase sensitivity of breath testing for SIBO (11), capturing the 20 to 30 percent of the general population who produce methane as a main byproduct of carbohydrate fermentation.

Carbohydrates such as lactulose and glucose are the most widely used substrates. Glucose testing is more specific, but less sensitive; in other words, it has a higher rate of false negatives and a lower rate of false positives. Lactulose testing is more sensitive, but less specific; it has a higher rate of false positives and a lower rate of false negatives (5). Gases produced from bacterial fermentation of these carbohydrates in the small intestine can be measured in the breath using gas chromatography as an indirect measure of bacterial load. The patient provides a baseline breath sample and then consumes the substrate (50g of glucose or 10g of lactulose and about 120 to 200 mL of water). Breath samples are collected every 20 minutes for a total of three hours, and both hydrogen and methane are measured at each time point.

Breath testing certainly has advantages, but it can also be subject to misinterpretation (12, 13). Breath testing is sometimes unable to distinguish small bowel from colonic substrate metabolism. This is particularly problematic for the substrates glycocholic acid, d-xylose, sorbitol, and lactulose because they are incompletely absorbed in the small intestine. Some researchers have also speculated that some bacteria that colonize the small intestine produce hydrogen sulfide rather than methane or hydrogen (14). Currently available breath tests do not test for hydrogen sulfide, but this may change in the future—which would further improve accuracy of breath testing.

Also, a study in 2015 proposed that overgrowth of fungus (small intestinal fungal overgrowth, or SIFO) in the small intestine may cause symptoms similar to those observed in SIBO. There is currently no commercially available test for SIFO, but hopefully this will become available soon (15).

Methodological Considerations for SIBO Breath Testing

Despite its limitations, hydrogen and methane breath testing remains the best option for diagnosing SIBO in clinical practice. The diagnostic accuracy of breath testing can be maximized by careful patient selection for testing, proper test preparation, and standardization of test performance, as well as test interpretation. In this section, we’ll explore some methodological considerations.

First, there is much controversy regarding an increased baseline breath hydrogen level. This can occur as a consequence of poor oral hygiene, recent smoking, or ongoing bacterial fermentation of poorly absorbed carbohydrates in the stomach, small intestine, or colon. This can be minimized by avoiding a diet rich in fermentable carbohydrates on the day before testing, and an overnight fast. There are a few other factors to consider leading up to the date of the test (16).

Here are my recommended test preparation steps to improve breath test accuracy:

  • Two weeks before the test: Ensure the patient finishes any antimicrobials (drugs, botanicals, or nutrients), is not having any atypical diarrhea, and will not receive a colonoscopy or barium enema before the test.
  • Four days before the test: Patient should avoid all laxatives, high-dose vitamin C, and magnesium supplements.
  • One to two days before the test: Patient should avoid all high-fiber and lactose-containing foods, condiments, spices, and herbs. Consume only meat, fish, plain steamed jasmine rice, eggs, and clear meat broth. Limit fat and oils, salt and pepper, and weak black coffee and tea. Stop all non-essential medication.
  • 12 hours before the test: Patient should begin a water fast and wake at least one hour prior to the test. The patient should avoid smoking and vigorous exercise before consuming the substrate.


To sum up, quantitative culture is largely impractical outside of a research setting. Hydrogen and methane breath testing remains the best option in clinical practice but has many caveats for accurate interpretation, and there is high risk for both false negatives and false positives. Several steps (outlined above) can be taken to help improve test accuracy.

For more on diagnosis, test interpretation, and treatment of SIBO, be sure to check out my SIBO seminar through the Kresser Institute for Functional and Evolutionary Medicine.


  1. For the two weeks prior to the test where you recommend stopping any microbials, do you include probiotics in that as well?