Differentiation between different etiologies of HUS is highly important regarding treatment and outcome. Since aHUS is a diagnosis per exclusionem, proving proof of STEC infection in a “typical” STEC-HUS is essential. However, fecal diagnostics, the gold standard to diagnose STEC-HUS, have some major drawbacks, most importantly due to the natural course of disease and low inoculums. Serological diagnostics, like using anti-O157 LPS antibodies, have proven its added value to fecal diagnostics, although this diagnostic assay has a different bottleneck: potential cross-reactivity and limited sensitivity. Here, we show that the novel glyco-iELISA to detect anti-O157 antibodies is highly sensitive and specific, and its use in STEC diagnostics leads to more patients displaying positive STEC-O157 infections causing HUS. More importantly, using glyco-iELISA STEC O157-infections could be detected for a long period of time after start of the disease.
With this study, the clinical utility of the glyco-iELISA was assessed. Melli et al. were the first to publish their findings regarding this novel glyco-iELISA using bacterial-engineered glycoproteins for serotype O157, O145, and O121 . In a cohort of 71 samples taken from pediatric patients (comprising both STEC-positive patients and STEC-negative patients with clinical suspicion of STEC-HUS), they showed that the glyco-iELISA was highly sensitive and specific. Furthermore, no cross-reactivity between the previous serotypes was observed, confirming our results. The same group published a second article in 2017 by Castillo et al. where they further investigated cross-reactivity between different STEC serotypes (resp. O111, O103, O45, O26, O104) and other gram-negative bacteria (salmonella, Brucella abortus, Yersinia enterocolitica O9). Again, no cross-reactivity was observed . This is in contrast to LPS-ELISA, where clear cross-reactivity between different serotypes has been reported . Overall, with access to the glycoproteins, glyco-iELISA is an easily implemented and performed assay with stable results.
Ideally, one would calculate sensitivity (proportion of patients with STEC-HUS in which the glyco-iELISA is positive) and specificity (proportion of patients with TMA caused by other conditions than STEC in which the glyco-iELISA remains negative) for an assay like glyco-iELISA. However, various problems arise when attempting to do so. The most prominent one has to do with the accuracy of the gold standard to diagnose STEC-HUS. Since fecal diagnostics are not sufficient to diagnose all STEC-HUS patients, no optimal gold standard is present to calculate sensitivity and specificity. Moreover, serology should not replace fecal diagnostics, but should be used in addition, to complement microbial diagnostics and broaden the time window to detect STEC. Hence, accurate estimation of sensitivity and specificity is not feasible. However, in our cohort, in contrast to LPS-iELISA, all patients with proven O157 in the feces were positive with glyco-iELISA, indicating high sensitivity (100% in our cohort). Furthermore, in patients with proven STEC infection with a non-O157 serotype, glyco-iELISA for O157 remained negative, again in contrast to LPS-ELISA for O157, indicating high specificity.
As stated previously, cross-reactivity between LPS of different gram-negative bacteria is a known problem due to the conserved lipid A part of the LPS molecule. We hypothesized that cross-reactivity, as observed in LPS-based ELISAs, can present as a false-positive test result in the O157 LPS-ELISA. This may be due to the presence of antibodies against other non-O157 STEC serotypes or even other gram-negative bacteria, which to some extent are able to bind to the lipid A part of STEC serotypes. Interestingly, using the highly specific and sensitive glyco-iELISA, we observed an increase in the detection of STEC infections, rather than a decrease due to false-positive results. Different explanations could explain this better performance. Primarily, STEC serotype O157 is still a highly prevalent serotype causing HUS in the Netherlands. Therefore, not much cross-reactivity could be found, since non-O157 serotypes causing HUS are less common. Furthermore, as shown in our single-center cohort, the glyco-iELISA is able to detect all patients with confirmed O157 serotype in the feces and remains negative in patients with a confirmed infection with other serotypes. Hence, the negative result obtained with the glyco-iELISA seemed accurate, indicating that the LPS-ELISA is probably a false-positive result due to cross-reactivity between different STEC serotypes, as was found to be the case in two of our patients. Other methods to detect serology have been reported, such as line blot immunoassay and immunoblotting, however, all use purified LPS to detect antibodies; hence, potential cross-reactivity remains present .
Interestingly, of the 52 samples of relatives without clinical HUS features, 10 (19.2%) had antibodies detected with the glyco-iELISA, indicating STEC O157 transmission person-to-person or intake of the same contaminated food. In all except one, the index patient tested positive for STEC infection. Although we have no clinical information about relatives in our study, we could show that family members of STEC-HUS patients with no or mild signs of gastrointestinal infection can develop antibodies against O157. These results are in line with Ludwig et al. who reported that 17% of the household contacts (symptomatic as well as asymptomatic) of STEC-HUS patients had LPS IgM antibodies against STEC serotype O157 . The exact rate of IgM antibodies against STEC serotypes in the healthy population is still unknown. However, to exclude potential false-positive results, one could consider only testing for the presence of IgM and not IgG, since IgG can be present for years after infection. Yet, we would recommend testing family members of patients with STEC (with fecal diagnostics and serology), especially in patients who tested negative for STEC infection. By providing proof of STEC infection in household contact, the diagnosis of STEC-HUS in the index patient despite negative diagnostics becomes more likely.
We found IgM antibodies against STEC O157 up to 55 days after onset of the disease. These results are in line with previous reported kinetics of IgM (LPS-based assay) against STEC by Chart et al. . Hence, in contrast to fecal diagnostics in which the isolation rate declines quickly after the initial symptoms (within 1 week), serology (both LPS- and glyco-iELISA) broadens the time window to diagnose STEC infections. Furthermore, when serum is collected too early in the course of the disease, serology could be negative due to as yet incomplete seroconversion, as was the case in two of our patients. In case of a negative serology result tested in serum collected within 7 days after disease onset, the advice would be to collect and test serum again after 7 days for a re-evaluation. As described previously, the added value of serology increases even more 7 days or more after the start of the symptoms .
Limitations of this study are the retrospective nature and the lack of clinical data of the national TMA cohort. Serological detection of STEC infection by detection of anti-O157 antibodies in serum is advised in the national guideline of diagnostic workup for TMA at presentation. Presumably, some patients in the national cohort had a different diagnosis that not only comprised STEC-HUS, but also aHUS or other causes of TMA. Concerning the single-center cohort of pediatric patients, STEC infection could not be detected in seven patients with clinical suspicion of HUS. Yet, aHUS as a diagnosis is highly unlikely regarding the clinical presentation with bloody diarrhea in all seven patients. Although aHUS can present in 30% of cases with gastrointestinal infection, bloody diarrhea is seldom reported in aHUS. Also, follow-up data showed no disease recurrence, making aHUS highly unlikely in this single-center cohort. In three patients, genetic analysis was performed and showed no pathogenic mutations in complement genes associated with aHUS. Furthermore, in most patients, serology was only tested at one time point. Seroconversion takes 3–5 days; hence, patients who were seen early in the course of disease could be false negative. Furthermore, we focused on the still most prevalent STEC serotype causing HUS in our country, O157; however, nowadays, non-O157 serotypes are increasingly detected as a cause of HUS. The relatively high number of patients with STEC O157 infection in our cohort could be explained by the substantial number of STEC-HUS patients who were included from the late 1990s, when serotype O157 was, as it still is, the main serotype to cause STEC-HUS. Nowadays, non-O157 serotypes causing HUS are increasingly detected, partly explained due to new and improved diagnostic assays. In this study, non-O157 STEC serotypes were not detected [2, 4]. It would be very worthwhile in the near future to examine the 14% of clinical STEC-HUS patients who were negative in fecal and serological diagnostics for other STEC serotypes with glyco-iELISA. Hence, future plans are to expand glyco-iELISA to detect multiple serotypes. Melli et al. have already described the use of glyco-iELISA for STEC serotypes O145 and O121, with comparable results regarding absence of cross-reactivity and sensitivity of the assays . Furthermore, it is highly important to differentiate between STEC-HUS and aHUS as soon as possible, to start appropriate treatment. Since the current glyco-iELISA takes at least 24 h to perform, future studies should focus on improving this assay for bedside use. For example, by using lateral flow technology, one could develop a point of care test for patients presenting with TMA.
In conclusion, serological assays for STEC O-antigens have a place in the diagnostic workup plan of patients with TMA. Moreover, since aHUS is a diagnosis per exclusionem, it is highly important to diagnose STEC-HUS. Therefore, we advocate always combining fecal diagnostics together with serological diagnostics to achieve optimal diagnostics and prevent unnecessary use of the highly expensive orphan drug eculizumab. The optimal assay to determine serological antibodies against STEC serotype O157 is the glyco-iELISA.