Clinical manifestations of NCGS may overlap with other conditions, such as irritable bowel syndrome (IBS), making diagnosis difficult. In a prospective multicenter study conducted by Volta et al.17 at 38 Italian centers, 486 patients with suspected NCGS were evaluated between 2012 and 2013. The symptoms identified included abdominal pain and distension, altered bowel transit (diarrhea or constipation), in addition to multiple extra-gastrointestinal symptoms including iron or folate deficiency anemia (Table 1).

Clinically, distinguishing NCGS from CD is complex, but an important difference is the time to onset of symptoms: in NCGS, it usually occurs between a few hours and one day after gluten consumption, while in CD this may take weeks or years.17 Biesiekierski et al.,1 in a clinical trial using a DBPC test in 34 patients with IBS without CD, found that IBS-like symptoms and fatigue, as isolated extraintestinal manifestation, recurred more frequently in the gluten-exposed group than in the placebo group (68% and 40%, respectively). Although IBS-type bowel symptoms are more common in patients with NCGS, they also usually report at least two extraintestinal symptoms, the most common being “brain fog” (feeling of lethargy after eating gluten) (42%) and fatigue (36%).6,18 These results have led some authors to consider that NCGS and IBS could be manifestations of the same disorder.15,19 However, other investigators emphasize that the NCGS shows no systemic symptoms in the IBS.18

Cárdenas-Torres et al.20 highlight key differences between NCGS and IBS. NCGS is triggered by gluten, ATI and FODMAPs, while IBS is not linked to specific components. Clinically, NCGS includes both intestinal and extraintestinal symptoms, while gastrointestinal symptoms predominate in IBS. Its diagnosis is based on a double-blind placebo-controlled (DBPC) trial, unlike IBS, which uses Rome IV criteria. NCGS treatment involves GFD and/or low FODMAPs, while IBS requires a comprehensive approach.

Diagnosis of NCGS should be considered in patients with persistent intestinal symptoms (such as those of IBS) and/or extraintestinal symptoms, in the absence of positive serological markers for CD and WA, which worsen after gluten intake. In 2014, an expert panel meeting in Salerno (Italy) proposed standardized diagnostic criteria in the absence of specific biomarkers. The most reliable method is a DBPC trial, which assesses the response to the elimination and introduction of gluten.3 The patient must identify one to three main symptoms, assessed using a numerical scale from 1 to 10, with a positive response being when there is a 30% change in at least one of these symptoms between phases with gluten and placebo.3,21,22 The protocol includes two phases: an elimination phase (at least six weeks on a GFD), followed by gluten or placebo challenge for one week each, with a one-week gluten-free interval. The gluten preparation should include 8 g daily, at least 0.3 g of ATI and be free of FODMAPs; the placebo should be indistinguishable in texture, taste and nutritional content.3 For a complete diagnostic evaluation, the patient should be eating gluten. If the patient already follows a GFD, only the challenge phase can be performed. In both phases, a self-administered questionnaire based on a modified version of the Gastrointestinal Symptom Rating Scale (GSRS) is used, which also includes extraintestinal symptoms.23 In clinical practice, evaluating extraintestinal manifestations such as anemia may facilitate the diagnosis of NCGS.14

Lionetti et al.,16 in a systematic review of clinical trials to diagnose NCGS, found only three studies that applied the Salerno criteria (Table 2). In these studies, 40% of patients relapsed on gluten versus 24% on placebo (p = 0.003),1,16,24,25 indicating a significant relationship between gluten and the occurrence of symptoms. The relative risk (RR) of relapse with gluten was also significant (2.8; p = 0.002). However, when considering all studies from the review, only 36% of patients relapsed with gluten versus 31% with placebo (p = 0.2; RR = 0.4; p = 0.16), with no statistically significant difference,16,26 In the absence of specific biomarkers, these data support a causal relationship between gluten and recurrent symptoms in NCGS patients when the Salerno criteria are used for diagnosis.16,21,27 However, despite being considered the “gold standard test”, this DBPC provocation test only identifies between 30% and 40% of patients with clinical suspicion of NCGS,16,26 suggesting that it may not capture the full heterogeneity of this condition. Studies such as those by Di Stefano et al., Elli et al. and Zanini et al. showed diagnosis rates after DBPC provocation of only 52%, 14% and 34%, respectively.28–31 However, according to Lionetti et al., these investigations did not strictly apply the Salerno criteria.16 On the other hand, this protocol has not been clinically validated, has a high risk of a nocebo effect (where patients experience symptoms with placebo, due to their negative expecations)1,17,30,32 and does not consider other components of wheat such as FODMAPs or ATIs, which could also cause symptoms in NCGS.33

Several observational studies have shown that NCGS may present immunological and systemic manifestations similar to those of CD. Volta et al.,17 after studying 486 patients with suspected NCGS, found a high association with IBS (47%), a significant prevalence of other food intolerances (1/3 of patients), and IgE-mediated allergies (20%). In addition, a high prevalence of autoimmune diseases such as Hashimoto thyroiditis and psoriasis and a high frequency of nickel allergy (as in CD) were observed. In agreement, Carroccio et al.34 found a higher frequency of anemia, autoimmune disorders, ANA positivity, and HLA-DQ2/DQ8 haplotypes in patients with NCGS or CD as compared to those in the IBS group.

As regards the serological profile, Volta et al.18 found that over 50% of patients with NCGS were positive for anti-gliadin IgG antibodies, but negative for specific CD markers: anti-endomysial antibodies (EmA), tissue transglutaminase (tTGA) or deamidated gliadin peptide antibodies (DGP-AGA). An increase in intraepithelial lymphocytes (IEL) was observed in 42% (Marsh 1), suggesting a possible mild alteration of the intestinal mucosa. Potential biomarkers for NCGS such as eosinophilic infiltration of the intestinal mucosa, increased CD3+ intraepithelial T cells, presence of mast cells, cytokine profiles, microRNA signatures, and RNA transcription patterns have also been proposed.20,33,35–40

In another study, Carroccio et al.32 analyzed 276 patients with NCWS, identifying two clinical subgroups, one with only NCWS and one with multiple food hypersensitivity. Both groups had a higher frequency of anemia, self-reported wheat intolerance, atopy, and childhood food allergies than the IBS group. In addition, patients with NCWS had eosinophilic infiltration of the intestinal mucosa and a high frequency of anti-gliadin antibodies and basophil activation in vitro. Those with only NCWS showed a similar clinical profile to that of CD, while those with multiple food sensitivity more closely resembled allergic patients. Approximately 50% have HLA-DQ2/DQ8 haplotypes.41

Finally, Cárdenas-Torres et al. and Roszkowska et al.20,42 compared NCGS, CD, and WA, showing that CD is an autoimmune disease with specific markers and intestinal atrophy (Marsh I–IV); NCGS, in contrast, involves innate immunity and mild lesions (Marsh 0–II); while WA is a mediated IgE reaction. Both NCGS and WA may present extraintestinal symptoms, but their diagnosis requires different approaches and does not present the immunological or histological markers characteristic of CD. Although the Salerno protocol represents the most rigorous method for the diagnosis of NCGS, its clinical applicability is limited, and diagnosis should therefore be supported by a comprehensive evaluation including gastrointestinal symptoms, systemic symptoms, and investigations.

Studies that used the Salerno criteria to diagnose NCGS showed variability in diagnosis rates after the DBPC provocation test, as determined by the design and population included. Rosinach et al. reported the highest rate (91%) in the group that ingested gluten (55.5% of the total sample). Shahbazkhani et al., with a sample of patients initially diagnosed with IBS, found a rate of 74.3% in the gluten group (31.1% of the total sample), while Biesiekierski et al. reported 68% in the gluten group (38.2% of the total sample). In the observational study by Barbaro et al., the diagnosis was confirmed in 55.1% of the 49 patients evaluated retrospectively under a modified version of the Salerno criteria. Finally, Mansueto et al. and Zanini et al. also used these criteria in previous stages to select their patients, also showing an association between anemia and NCGS.1,14,24,25,38,40

Overall, the included studies exploring the prevalence of anemia in patients with NCGS show a relevant but poorly recognized clinical association. This may be associated with mild inflammation of the small intestine (intraepithelial lymphocytosis — Marsh 1)14,17,18,38,40,46–48 or by eosinophilic infiltration.32,40,46,47 Or due to impaired iron absorption, even in the absence of the structural damage typical of CD. In general, these studies agree that anemia is more common in patients with NCGS than in those with IBS, and in some cases comparable to that seen in CD.

This study highlights a significant heterogeneity in the definition and evaluation of anemia among the studies included. Although many studies use the hemoglobin threshold of <12 g/dL as the criterion, others do not specify or differentiate between types of anemia. In addition, there is variation in the hematological markers used, from hemoglobin only to ferritin, serum iron, transferrin, folate or vitamin B12. This lack of standardization, together with the methodological diversity of the studies (retrospective, prospective, clinical trials with or without control group), makes comparison of results difficult and excludes the possibility of performing a meta-analysis. There was also a predominance of European studies, especially Italian studies, which limits the generalisation of the findings. Only 6 of 18 studies applied the Salerno diagnostic criteria, and several lacked a control group, and selection and publication bias may be present, with an under-representation of studies with negative results.