Diagnosing celiac disease in patients with a history of lymphoma: factors that matter
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- Balihar, K., Kozeluhova, J., Hejda, V. et al. Wien Klin Wochenschr (2013) 125: 696. doi:10.1007/s00508-013-0437-9
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The aim of this study was to identify pitfalls in establishing the diagnosis of celiac disease (CD) in patients with a history of lymphoma.
A total of 103 patients with a history of lymphoma had anti-tissue transglutaminase antibodies (atTGA) and their class A, G, and M immunoglobulin (IgA, IgG) levels determined. Patients with atTGA positivity underwent enterobiopsy and CD-associated HLA locus testing.
The mean age of patients was 55 ( ± 13.5) years. The predominant lymphoma types included B-type non-Hodgkin’s lymphoma (B-NHL, 66 %), T-type NHL (8 %), and Hodgkin’s lymphoma (26 %). Serological positivity was documented in 3.9 % of cases; one patient had the diagnosis of CD confirmed by enterobiopsy. In 11 patients (10.7 %), IgA levels were decreased to a various extent; of these patients, 10 were shown to have also their IgG levels decreased. The median time from follow-up to blood collection was 58 (32–104) months. The decrease in immunoglobulin levels correlated with a more advanced stage of the tumor (Ann Arbor III–IV) at the time of diagnosis [1.4 (0.9–2.0) g/l versus 2.4 (1.5–3.0) g/l for IgA, p = 0.0001; and 9.4 (7.2–11.5) g/l versus 11.2 (10.3–12.3) g/l for IgG, p = 0.001] and older age [65 (54–72) years versus 55 (44–61) years for IgA, p = 0.04; and 69 (59–74) years versus 53 (43–61) years for IgG, p = 0.0001]. Rituximab therapy in B-NHL patients had no effect on the subsequent incidence of decreased IgA levels.
Reduced IgA and IgG levels represent important factors contributing to the low detection rate of serological screening for CD in patients with a history of lymphoma.
KeywordsCeliac diseaseNon-Hodgkin’s lymphomaAnti-tissue transglutaminase antibodiesAutoimmunityImmunoglobulin deficiency
Diagnose einer Zöliakie bei Patienten mit Lymphom in der Anamnese – wichtige, die Diagnose beeinflussende Faktoren
Identifikation von Fehlermöglichkeiten bei der Diagnosestellung einer Zöliakie bei Patienten mit Lymphom in der Anamnese.
Es wurde bei insgesamt 103 Patienten mit einem Lymphom in der Anamnese eine Bestimmung der Anti-Gewebs Transglutaminase Antikörper (atTGA) und deren Klasse A, G, und M Immunglobulin (IgA, IgG) Konzentrationen durchgeführt. Patienten mit positivem atTGA Nachweis wurden einer Darmbiopsie unterzogen. Außerdem wurden sie auf Vorliegen des Zöliakie-assoziierten HLA-Lokus untersucht.
Das mittlere Alter der Patienten lag bei 55 Jahren ( ± 13,5). Die Patienten hatten folgende Lymphomtypen: B-Typ Non-Hodgkin Lymphom (B-NHL) 66 %, T-Typ NHL 8 %, und Morbus Hodgkin: 26 %. Bei 3,9 % der Patienten wurde ein positiver Test im Serum erhoben. Bei einem Patienten wurde die Diagnose durch Darmbiopsie bestätigt. Bei 11 (10,7 %) Patienten waren die IgA Konzentrationen in verschiedenem Ausmaß erniedrigt; 10 dieser Patienten hatten auch erniedrigte IgG Spiegel. Die mediane Zeit vom Follow-up zur Blutabnahme war 58 Monate (32–104). Der Abfall der Immunglobuline korrelierte mit dem Ausmaß des Fortschreitens des Tumorstadiums (Ann Arbor III–IV) zum Zeitpunkt der Diagnose [IgA 1,4 (0,9–2,0) g/l versus 2,4 (1,5–3,0) g/l; p = 0,0001 und IgG 9,4 (7,2–11,5) g/l versus 11,2 (10,3–12,3) g/l; p = 0,001], und mit dem zunehmenden Alter [65 (54–72) Jahre versus 55 (44–61) Jahre für IgA; p = 0,04 and 69 (59–74) Jahre versus 53 (43–61) Jahre für IgG; p = 0,0001]. Eine Rituximab Therapie bei Patienten mit B-NHL hatte keine Auswirkung auf die folgende Inzidenz erniedrigter IgA Konzentrationen.
Erniedrigte IgA und IgG Spiegel sind ein wesentlicher Faktor zur Erklärung der niedrigen Detektionsrate beim serologischen Screening auf Zöliakie bei Patienten mit einem Lymphom in der Anamnese.
SchlüsselwörterZöliakieNon-Hodgkin LymphomAnti-gewebs Transglutaminase AntikörperAutoimmunitätImmunglobulinmangel
Anti-tissue transglutaminase antibody IgA
Anti-endomysial antibody IgA
B-cell non-Hodgkin’s lymphoma
T-cell non-Hodgkin’s lymphoma
Chronic lymphocytic leukemia
Celiac disease (CD) is an autoimmune enteropathy triggered by dietary gluten in genetically predisposed individuals. The prevalence of CD in Europe is reported to be in the range of 0.3–2.4 % . The disease often goes unrecognized because of the large heterogenity of its clinical features, which are predominantly extraintestinal and often very subtle in adulthood. Targeted screening for CD is indicated with a variety of extraintestinal symptoms (sideropenic anemia, premature osteoporosis, impaired fertility, etc.) and associated autoimmune diseases (autoimmune thyroiditis, type-1 diabetes mellitus, systemic connective tissue diseases, etc.) . The first diagnostic tools recommended for CD screening in the general population include anti-tissue transglutaminase antibody immunoglobulin class A (IgA; atTGA) and anti-tissue endomysial antibody (aEMA) . Any IgA deficiency more common in CD patients than in the non-CD population decreases the diagnostic yield, and if confirmed, it is recommended to perform screening for class G antibodies [3, 4]. In patients with a deficiency of both immunoglobulins, the benefit of serology for the diagnosis of CD is limited or reduced almost to zero.
Malignancies are a major complication of untreated or undiagnosed CD. The association between CD and presence of lymphoma has been investigated in a number of studies, and it is now generally recognized that CD patients not adhering to gluten-free diet are at a higher risk for developing lymphoma during the course of their disease, although the risk is not as high as previously thought . In a recent, and to date the largest, study designed to investigate the incidence of CD in patients with first-ever diagnosed non-Hodgkin’s lymphoma (NHL), the risk ratio for developing NHL compared with the general population was estimated at 2.7 . It was particularly on the basis of the results of that study that routine CD screening in individuals when first diagnosed to have lymphoma was not—unlike the aforementioned conditions—recommended because the detection rate of such a screening program would be relatively low, although this conclusion has not yet been incorporated into the current guidelines [2, 7].
Still, there are a variety of recommended indications for CD screening, and conceivably, the indication may be extended to include the population of patients with a history of lymphoma and long-term survival after anti-cancer therapy. The levels of immunoglobulins in pediatric patients with acute leukemia have been shown to decline to values as low as 50 % of baseline and not returning to normal levels before 1 year . Although no data from long-term follow-up for adults are available, it can be reasonably assumed that restoration of humoral immunity functions in the older-age population will take a longer time to occur. The present study seeks to characterize patients with a history of lymphoma, and to identify the pitfalls of diagnosis of CD in this patient population.
Patients and methods
The study was performed in accordance with the ethical principles of the Declaration of Helsinki, and was approved by the local research and ethics committee. All patients signed inform consent.
Included into the study were patients with diagnosed lymphoma receiving treatment at the Department of Hematology and Oncology of the Teaching Hospital in Pilsen from April 1, 2009, through March 31, 2010. Exclusion criteria were defined as failure to sign informed consent, age below 18 years, previous allogeneic bone marrow transplantation, immunosuppressive therapy within 6 months before blood collection, or continued anti-lymphoma therapy over the past 6 months before baseline examination. Overall, 157 patients treated for lymphoma had been selected to participate in the study, of which 54 did not meet the inclusion criteria and were excluded. As a result, data of 103 patients with a mean age of 55 ( ± 13.5) years, with an even male-to-female ratio (women, 52 %), were analyzed. Individual lymphomas were classified using the World Health Organization system ; the clinical stage of the tumor at the time of obtaining the biopsy sample was determined according to the 2006 Revised Response Criteria of the American Society of Clinical Oncology . Tumor staging at the time of diagnosis was performed using the Ann Arbor staging system, and two patients with chronic lymphocytic leukemia (CLL) were assessed using the Rai classification .
Serum atTGA detection was performed by immunometry using a commercially available IMMULITE 2000 anti-tissue transglutaminase IgA kit (Siemens Healthcare Diagnostics, Llanberis, UK). The declared analytical sensitivity of the kit is 1.32 U/ml. Calibration was performed using commercially available calibration solutions with known atTGA levels; results greater than 4 U/ml were defined as positive.
Determination of total immunoglobulin levels
Quantitative determination of serum IgA, immunoglobulin class G (IgG), and immunoglobulin class M (IgM) levels was performed by immunonephelometry using a Behring nephelometer II device (Siemens Healthcare Diagnostics GmbH, Marburg, Germany). Evaluation was performed by comparing the results with known levels of a commercially available standard. The defined reference ranges are 0.7–4 g/l for IgA, 7–16 g/l for IgG, and 0.4–2.3 g/l for IgM.
Small bowel biopsy
During endoscopy, four mucosal specimens were obtained from various sites of the second duodenal segment. Tissues for light microscopy were immediately oriented using a stereomicroscope, fixed in 4 % formaldehyde, and subsequently embedded in paraffin using routine procedures. Five-micrometer-thick sections were cut from the tissue blocks and stained with hematoxylin and eosin and with periodic acid-Schiff reaction. The following primary antibodies were used for immunohistochemical detection and further classification of intraepithelial lymphocytes: CD3 (clone PS1, 1 : 200, Novocastra, Newcastle upon Tyne, UK) and CD8 (clone C8/144B, 1 : 50, Dako, Glostrup, Denmark). The primary antibodies were visualized using the supersensitive streptavidin-biotin-peroxidase complex (Biogenex, San Ramon, CA). Appropriate control slides were used as recommended by suppliers. The histological patterns of mucosal samples were classified according to the Marsh–Oberhuber classification.
Patients with serum atTGA positivity underwent genotyping for CD-associated HLA loci using polymerase chain reaction–sequence-specific primer using a commercially available HLA-READY GENE Coeliac Disease kit (Inno-Train Diagnostik GmbH, Kronberg, Germany), detecting the following alleles: DQB1*02 : 01, 02 : 02, and 03 : 02, and DQA1* 05 : 01 and 05 : 05.
All patients meeting the inclusion criteria had their atTGA and total IgA, IgG, and IgM levels detected. Screening of patients with increased atTGA levels was complemented with enterobiopsy and CD-associated HLA locus testing. Also recorded were patient baseline demographic characteristics, lymphoma class, primary lymphoma localization, clinical status of the patient, staging at the time of lymphoma diagnosis, previous lymphoma treatment characteristics, and the interval since the last chemotherapy.
Statistical analysis was performed using the Kolmogorov–Smirnov test, c2 test, Mann–Whitney test, Spearman correlation rank test, Friedman test, and analysis of multivariance. These tests are reported with individual results; their values were considered significant if exceeding 95 % level of significance.
None of the 103 patients with analyzed data had a history of CD. A total of 68 patients had B-type NHL (B-NHL; 66 %); there were 8 T-type NHL (T-NHL) cases (8 %); and 27 individuals had Hodgkin’s lymphoma (HL; 26 %). No enteropathy-associated T-cell lymphoma was identified among T-NHL patients. At the time of inclusion into the study, all patients with T-NHL and HL were in complete remission, two B-NHL group patients were in partial remission, two showed mild disease progression (without indication to the beginning of therapy at the time of inclusion), and one female patient with CLL had not been treated since diagnosis (Table 1). Nodal and extranodal lymphoma localizations at the time of diagnosis were equally frequent (nodal localization, 52 %). The extranodal forms were predominantly B-NHL (82 %) being present in bone marrow (12), multiple organs, primarily non-gastrointestinal localization (8), spleen (6), tonsils (4), lungs (3), salivary gland (3), soft tissue (2), and thyroid (2). There were nine primary gastrointestinal lymphomas (three in stomach, one in ileum, four in liver, and one in oral cavity). At the time of diagnosis, less advanced stage tumors (Ann Arbor I–II) were slightly predominant (56 %; Table 2).
Characteristics of lymphoma patients
Type of lymphoma
Time since ChT (months)
68 (66 %)
28 + /40−
Diffuse giant-cell lymphoma
Mantle cell lymphoma
Chronic lymphatic leukemia
Marginal zone lymphoma
8 (8 %)
Peripheral T lymphoma
Anaplastic giant-cell lymphoma
27 (26 %)
Lymphomas by localization
54 (52 %)
23 (43 %)
28 (51 %)
3 (6 %)
49 (48 %)
4 (8 %)
40 (82 %)
5 (10 %)
40 (39 %)
9 (9 %)
Ann Arbor Stagesa
Previous lymphoma treatment
Three patients with B-NHL were not treated with chemotherapy (two patients were treated with local radiotherapy, one patient untreated). Of 100 patients treated with chemotherapy, 50 received CHOP-like chemotherapy (cyclophosphamide, adriamycin, vincristine, and prednisone) with or without rituximab, 12 got fludarabine-based regimes, 12 were administered intensive induction chemotherapy, 23 (mostly HL) got BVD/CEVD regimes (adriamycine, bleomycin, vinblastine, and dacarbazine/lomustine, etoposide, vinblastine, and dexamethasone), and 3 were administered chlorambucil-based regime. According to the type of the treatment, the patients were divided into two groups: those treated with intensified chemotherapy followed or not by autologous bone marrow transplantation (22 patients) and those treated with standard protocols (78 patients). All HL patients were given standard chemotherapy regimes. In contrast, almost all T-NHL patients were treated with intensified chemotherapy, and 12 B-NHL patients were given intensive regimes as mentioned earlier in the text. An average number of treatment cycles irrespective of diagnosis and treatment regimes was 5.5 ( ± 1.8). The median time since the last chemotherapy was 58 (32–104) months, with no difference between HL and NHL groups [73 (46–114) months versus 52 (28–88) months, respectively; p = 0.08], whereas T-NHL patients were on follow-up longer than patients with B-NHL [113 (74–139) months versus 49 (26–75) months, respectively; p = 0.02] (Table 1). The median follow-up in patients with decreased IgA and IgG levels was 34 (23.0–52.5) months.
Serological atTGA positivity
Four individuals were identified as atTGA positive (3.9 %). They were all women with a history of diffuse large-cell lymphoma, who were in complete remission. One of these patients refused to undergo subsequent enterobiopsy; however, HLA typing for CD-associated alleles proved negative, and the patient is unlikely to have CD. Although HLA typing in the remaining three patients showed CD-associated allele positivity, histological alterations in an enterobiopsy sample compatible with the diagnosis of CD were demonstrated in only one of them (1 %; Table 3). None of the three patients with atTGA positivity and CD-associated HLA loci was clinically symptomatic (including atypical presentations) or had another potentially CD-associated disease. A comparison of NHL and HL patients with those having non-gastrointestinal and gastrointestinal lymphoma localizations revealed no statistical difference in the incidence of atTGA positivity. Multivariate analysis showed that patients with increased atTGA levels were older [71 (63–79) years versus 55 (45–62) years, p = 0.02] and had higher IgA levels [3.1 (2.5–3.8) g/l versus 1.6 (1.2–2.6) g/l, p = 0.03]; no significant differences were found in the other parameters.
Patients with atTGA positivity
Ann Arbor stage
Time since ChT (months)
IgA (0.8–3.5 g/l)
Diagnosis of CD
DQA1 + DQB1 +
DQA1 + DQB1 +
Eleven patients showed various degrees of IgA deficiency (10.7 %), of which 10 had also decreased IgG levels (91 %). Values less than 50 % of the lower limit of normal were documented in five patients (4.9 %), whereas two patients (1.9 %) had very severe deficiency of all three followed immunoglobulin classes, compatible with diagnosis of common variable immunodeficiency. Lower levels of IgG were noted in 16 individuals (15.5 %) and IgM in 14 patients (13.6 %; Table 4). Patients with decreased IgA and IgG levels were older than those with normal values [65 (54–72) years versus 55 (44–61) years, p = 0.04, and 69 (59–74) years versus 53 (43–61) years, p = 0.0001, respectively]. NHL patients were shown to have lower IgA levels than HL patients (borderline significance, p = 0.05), with a statistical significance level higher for B-NHL versus HL (p = 0.02, Mann–Whitney test). Patients with more advanced lymphomas at the time of diagnosis (Ann Arbor III and IV) had lower IgA levels compared with patients with less advanced lymphomas [1.4 (0.9–2.0) g/l versus 2.4 (1.5–3.0) g/l, respectively; p = 0.0001]; a similar association was demonstrated for IgG levels [9.4 (7.2–11.5) g/l versus 11.2 (10.3–12.3) g/l, p = 0.001 (Mann–Whitney test)]. Twenty-eight B-NHL patients (41 %) received therapy with rituximab (a monoclonal antibody against B-lymphocyte transmembrane antigen CD20), with no correlation to IgA levels between treated and untreated patients. No correlation was found between the type of chemotherapy and plasma levels of IgA across the diagnoses of HL and NHL. Similarly, there was no correlation between the number of treatment cycles and IgA levels (Spearman correlation test and Friedman test). Finally, no correlation between follow-up duration and IgA levels was shown (Spearman correlation test).
Patients with decreased immunoglobulin levels
Type of lymphoma
Time since ChT (months)
IgA (0.8–3.5 g/l)
IgG (8.5–15.5 g/l)
Decreased immunoglobulin levels
An important factor affecting the degree of serological positivity in CD screening is the presence of IgA deficiency leading potentially to underestimation . In the general population, selective IgA deficiency is the most rare occurrence (0.11 %), and studies in patients after their lymphoma had been first detected reported minimal or no IgA deficiency [4, 12–14]. Regrettably, the largest study addressing this issue was not designed to document decreased IgA levels or IgA deficiency . Our study showed decreased IgA levels in 10.7 % and IgA deficiency in 1.9 % of patients, i.e., relatively high figures. Given the aforementioned data and prevalence in the general population, the decrease in IgA levels seen in our patients is most likely secondary to previous anti-lymphoma therapy, although IgA levels before onset of disease had not been recorded in our study. Importantly, the fact that most patients with decreased IgA levels showed also decreased IgG levels may have potentially had an adverse effect on IgG CD-antibody detection. The decrease in IgA levels correlated with age and an advanced tumor stage at the time of diagnosis; however, no patient with advanced lymphoma was shown to be atTGA positive. It can be reasonably assumed that patients with advanced lymphomas had more altered humoral component of their immune system, probably due to the disease itself rather than as a result of chemotherapy. This reasoning is supported by the absence of any correlation between the type and number of chemotherapy cycles and IgA levels. In addition, the older the patients, the lower are their abilities to maintain immunoglobulin production and form antibodies after anti-cancer therapy. The alteration of humoral immunity persisted in our patients for a long time. The median follow-up on therapy completion among patients with decreased IgA and IgG levels was 34 months, being 58 months for the whole group of our patients. The difference was not significant given the lack of literary data regarding the behavior of immunoglobulin levels in adults undergoing anti-cancer therapy over time. The long-term nature of these changes has only been suggested by some studies in pediatric patients as mentioned earlier in the text . Another factor potentially modulating immunoglobulin production is rituximab. It is a mouse/human chimeric IgG(1)-kappa monoclonal antibody that targets the CD20 antigen found on the surface of malignant and normal B lymphocytes, which is used in the treatment of CD20-positive B-NHL. Although treatment with rituximab induces lymphopenia in most patients, which typically lasts approximately 6 months, full recovery of B lymphocytes in peripheral blood is usually seen 9–12 months after therapy . In our study, rituximab therapy in B-NHL patients did not correlate with the presence of humoral deficiency, a finding indirectly suggesting reversibility of changes occurring in the immune system after this particular therapy.
The correlation between atTGA positivity and increased IgA levels, and the absence of atTGA positivity in patients with decreased IgA levels, supports the assumption that decreased IgA levels may adversely affect the detection rates of CD-specific antibodies, resulting in false-negative results. Although B-NHL patients showed lower IgA levels compared with HL patients, it was just the former where all cases of atTGA positivity were found. Consequently, it can be speculated that the actual incidence of CD could be somewhat higher—particularly in the B-NHL population—than documented by serological screening.
From the point of view of clinical practice, a deficiency in humoral immunity may not pose a problem only as regards detection of autoimmune diseases. Patients with a primary deficiency of humoral immunity have been shown to have an altered ability to form antibodies after vaccination against seasonal influenza as do patients after anti-cancer therapy [16, 17]. Based on our data, a similar problem can be expected in a proportion of patients with a history of lymphoma.
The 103 patients with a history of lymphoma in our study are representative of the long-term survivors of anti-lymphoma therapy without chronic immunosuppressive therapy, and do not suggest the incidence of lymphoma as in studies investigating the co-existence of CD and lymphoma at the time of diagnosis [6, 12, 13, 18]. In our study, atTGA positivity was chosen as a screening test preferred in the available guidelines for the general population despite the risk for more frequent positivity compared with aEMA [2, 13]. In four of our patients, the increase in atTGA values was relatively small, in the order of a triple of the norm. Although the female patient diagnosed to have CD had only a mild histological finding (Marsh 1), CD-associated HLA allele typing documented positivity in several loci, supporting the diagnosis of silent form of CD in this patient . In one case, the false positivity of the atTGA was confirmed by negative HLA typing. The remaining two patients with positive serology and HLA typing and normal enterobiopsy were classified as having potential CD in accordance with the Oslo nomenclature . The potential CD is not entirely rare in clinical practice, occurring in 6–32 % of healthy subjects who were tested for CD and had positive autoantibodies [1, 21]. Over time, the antibodies may disappear or, conversely, patients may develop clinically significant form of CD . None of our patients developed clinical signs of CD nor did they show increased atTGA levels during the subsequent 4-year follow-up. For this reason, none of these patients underwent re-biopsy.
However, the detection rate of such screening can be viewed as low and truly representative. Compared with the largest Czech study in 1,312 blood donors, serological positivity in our patient series is lower (7 % versus 3.9 %), potentially suggesting that decreased immunoglobulin levels do play a role in antibody detection . However, several facts should be taken into account in this context. First, the data sets were obtained from two different patient groups; second, cases of serological positivity determined in the Czech study were not confirmed by histology, and hence, the incidence of CD in the Czech population remains unknown; third, atTGA was determined using a test with a porcine transglutaminase antigen and not one with a human recombinant antigen, thus producing discordant results, as documented by Carrocio et al. . It is for the aforementioned reasons that no definitive conclusions can be made. The main limitation of our study is the small number of patients enrolled in only one center.
In conclusion, the rate of detection of CD in patients with a history of lymphoma is low and is consistent with the estimated prevalence in the general population, as reported in relevant studies . In agreement with earlier trials, screening for CD cannot be recommended in patients with lymphoma on a routine basis, but specifically in those with at least one other potentially associated disease or symptom. The main finding of the present study is the relatively frequent decreases in IgA and IgG levels in this patient population. Given the relatively low incidence of humoral deficiencies in the general population, these seem to be secondary to previous anti-cancer therapy and are most likely long-term, thus potentially underestimating the results of serological screening for CD and, eventually, also those of screening for other antibodies, primarily in patients with advanced lymphoma at the time of diagnosis and in elderly patients. In clinical practice, when screening for CD-specific antibodies, it is critical to rule out a deficiency of immunoglobulins in the class of antibodies being tested. In cases of suspected symptomatic CD, it is appropriate to perform enterobiopsy or, alternatively, to establish the diagnosis based on the high negative predictive value of CD-associated allele HLA typing.
Balihar K designed the study, performed the endoscopic diagnoses, and wrote the manuscript; Lysak D co-designed this study and coordinated patient enrollment into the study and was involved in editing the manuscript; Kozeluhova J and Hejda V were also involved in designing the study and editing the manuscript; Krcma M performed statistical analysis and was involved in editing the manuscript; Gorcikova J co-designed the study and performed the immunological analysis; Hes O performed histological examination of specimens and edited part of the manuscript; Matejovic M obtained research funding, supervised and coordinated the study, and finalized the manuscript.
This research was supported by the Charles University Research Fund (project number P36) and by the project Ministry of Health, Czech Republic, for conceptual development of research organization 00669806—Faculty Hospital in Pilsen, Czech Republic.
Conflicts of interest
All authors (Karel Balihar, Jana Kozeluhova, Vaclav Hejda, Michal Krcma, Daniel Lysak, Jitka Gorcikova, Ondrej Hes, and Martin Matejovic) of the above study declare that they have no conflict of interest.