Small bowel histology is still considered the gold standard for diagnosing CD, notwithstanding the fact that the morphological features are not specific, and that other conditions can produce similar findings[1,2
]. The possibility of a noninvasive diagnostic algorithm for CD has been explored before[16-19
], but no definitive standard has been established yet. Our first aim was to assess the diagnostic performance of serological tests in two patient groups with different risk levels of having CD. In this context, we postulated that different assays might perform differently in populations with low pre-test risk for the disease compared to those with high-risk. We hypothesized that this could change the selection of the best serological algorithm to be used in case finding among disorders with increased prevalence of CD (e.g. chronic anemia, osteoporosis, irritable bowel syndrome, etc
.) or for screening the general population. Notably, the use of serological tests for the selection process of cases in clinical situations with low pretest probabilities has been mostly based on the performance of assays assessed in cohort studies with post-test probabilities greater than 95%.
Considering the high-risk group, we confirmed that all the individual serological assays had very high diagnostic efficacy. Interestingly, our present study shows the DGP/tTG Screen test is the only assay with optimal sensitivity, and only the IgG a-DGP test had 100% specificity and PPV at the cut-off provided by the manufacturer. If the cut-off values were set to obtain 100% PPV, the sensitivity would be minimally reduced for the IgA a-DGP and the DGP/tTG Screen, but more profoundly affected for IgA a-tTG, IgG a-DGP and DGP Dual. The sensitivity for AAA would be reduced from 87.3% to 52.4% with the 100% PPV cut-off, making its use non recommendable in the diagnostic work-up. Therefore, our results highlight the value of the new DGP/tTG Screen, which was the most sensitive assay in detecting CD among subjects with high-risk for the disorder at both cut-offs: the value set by the manufacturers and at a 100% PPV. As far as we know, this is the second study showing the efficacy of the newly developed DGP/tTG Screen test for CD[26
As we expected, the serological tests did not perform as well in the low-risk group. The sensitivity of individual tests varied between 52.9% and 82.3%, and the highest were again the DGP/tTG Screen and the IgA a-DGP assays. The specificity was high, ranging from 88.2% to 99.0%. As expected, the NPVs were excellent (98.3% to 99.4%) and the PPVs were disappointingly low for all the assays, ranging from 17.6% for the IgA AAA test to 70.6% for the IgG a-DGP. The commonly used IgA a-tTG assay had an unacceptable PPV of 50.0%. Once again, we assessed the performance of individual assays at cut-off values that would result in a 100% PPV. Sensitivity dropped significantly to unacceptable values, with the highest being the DGP/tTG Screen at 64.7%. Notably, the sensitivity of the commonly used IgA a-tTG was 35.3%. Overall, these observations for patients with a low-risk for CD (with a prevalence that is intermediate between those disorders at risk and that of the general population) suggest the possibility of underdiagnosis using the most commonly employed serological algorithms. Interestingly, the sensitivity of the assays in the low-risk group was affected by the fact that three of the 17 new patients with villous atrophy (17%) had a minor damage (type IIIa) and were negative for all tests. These cases were considered as having a CD-like enteropathy. We confirmed former observations that a minor degree of damage is frequently detected in patients diagnosed from populations with low pretest probability[27
]. Furthermore, our observations on the behavior of serological tests in this group are consistent with former findings showing that CD patients with a minor histological damage might have seronegative results[21,22,25
]. However, confirmation of a definitive diagnosis of CD in seronegative cases (and even more in cases with lesser degree of histological damage) requires additional features indicative of gluten dependency that often are difficult to meet. This was the case with the small group identified in this study. Interestingly, one patient had a positive clinical response to the GFD, but was negative for the HLA-DQ2 and DQ8 investigated in the β1 chain. The other two cases died some time after the biopsy without having performed a GFD, one due to a myocardial infarct, and the other due to an esophageal malignancy diagnosed at the time of endoscopy. We estimate that the lack of the specific HLA alleles in the first case minimized the possibility of the patient having a gluten-triggered enteropathy[28
]. However, although the inclusion of these three not well defined cases has a negative impact on the performance of serology in the low-risk population, the fact of all patients in our study have had a biopsy evaluation, makes our study a reflection of real clinical practice. We consider that although the diagnosis of these cases is uncertain, they should be included in the analysis, unlike those cases with mild inflammation (Marsh’s type I) with a positive serology, which were excluded on the bases of our strict protocol.
To determine if a combination of assays could improve diagnostic accuracy, we explored the performance of all possible combinations of two serological tests, with the condition that a given combination was considered positive or negative if both assays were concordantly above or below the cut-off values, respectively. We observed that the performance of all combinations for the high-risk group was slightly lower than that of single assays, as evidenced by the AU ROC (> 0.960). However, the specificity and PPVs increased to 100% with acceptable sensitivity (above 90.5%) for all possible combinations excluding from this analysis AAA. Furthermore, we observed that combinations of the DGP/tTG Screen with either IgA a-tTG or IgA a-DGP add accuracy to the diagnosis or exclusion of CD.
In the low-risk group, all combinations had poorer performances than the single assays due to a lower sensitivity with minimally increased specificity. However, PPVs improved significantly (with most combinations approaching 100.0%) as expected. Once again, as it was for the high-risk population, the DGP/tTG Screen assay used in combination with the IgA a-DGP exhibited the best performance with acceptable sensitivity (82.3%), optimal specificity, and predictive values. Our observations are in agreement with a recent study from our group that clearly showed that the use of the DGP/tTG Screen assay enhances the sensitivity of detecting gluten sensitivity among a-tTG seronegative patients with CD-like enteropathy (including cases with dermatitis herpetiformis)[29
]. Interestingly, it is well-established in clinical practice to use the IgA a-tTG test to select patients for biopsy, in both case-finding processes and in population screening studies. The performance of this popular assay in the low pretest population suggests that its use alone does not seem to be a wise strategy, because it would miss up to 23% of potential new cases.
Based on the present findings, we finally analyzed the number of cases missed or biopsy procedures avoided in the theoretical situation where serology could be used as a single non-invasive tool for diagnosing CD. With this aim, we assessed the effectiveness of two algorithms for the use of a single assay or two-assay combinations. The algorithm for single assays was devised such that biopsy should only be performed for patients with a positive test, as the pretest risk was below 50% in most clinical situations and the number of biopsies avoided would be greater. The second algorithm was designed for combinations of two serological assays, in which biopsy would be omitted if a patients had two positive or negative assays. Biopsy would be reserved for patients with conflicting results.
For the high-risk group, the DGP/tTG Screen assay was the only single test that did not miss any CD cases, and would avoid duodenal biopsy in 56.5% of subjects. In contrast, the algorithm exploring combinations of two assays was highly effective, and could avoid intestinal biopsy in more than 92% of subjects. The higher performance was seen in the combinations using DGP/tTG Screen with other assays. As expected, the serological algorithms did less well in the low-risk group, as three cases were negatives in all assays. In this population, the two diagnostic algorithms did not differ significantly in terms of false negatives or biopsies avoided. The use of the DGP/tTG Screen and the IgA anti-DGP assays alone, in combination with each other, or in combination with other assays, would miss the three mentioned cases and avoid biopsy in 91.3% to 95.4% of subjects.
In conclusion, we suggest that appropriate use of CD serology might accurately identify the vast majority of CD patients in populations with different pretest probabilities. Furthermore, a negative serology might still miss underlying CD, but the clinical importance of the disease in such patients is probably minimal. The combination of two assays makes diagnostic accuracy higher. However, a proportion of patients (17%) in the low-risk group would be missed by all serology tests or their combinations. Definitive confirmation of CD in these seronegative cases is often difficult and doubtful, sometimes requiring long-term observation. We also confirm the additional value of the new DGP/tTG Screen assay, which resulted in a surplus to more conventional assays and should be considered as the best initial test in investigation for CD. Our study also suggests that this assay in combination with IgA a-tTG or IgA a-DGP could be used to obviate the need for duodenal biopsy in more than 92% of individuals in the high- and low-risk populations. Future validation of the algorithms is required to confirm our findings before new diagnostic guidelines are proposed.