RESEARCH ARTICLE

The Impact of Azathioprine-Associated

Lymphopenia on the Onset of Opportunistic
Infections in Patients with Inflammatory
Bowel Disease
Marius Vögelin1, Luc Biedermann1, Pascal Frei1, Stephan R. Vavricka1,2,3, Sylvie Scharl1,
Jonas Zeitz1, Michael C. Sulz1,4, Michael Fried1,2, Gerhard Rogler1,2, Michael Scharl1,2*
1 Division of Gastroenterology and Hepatology, University Hospital Zurich, University of Zurich, Zurich,
Switzerland, 2 Division of Gastroenterology and Hepatology, Stadtspital Triemli, Zurich, Switzerland,
3 Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland, 4 Division of
a11111 Gastroenterology and Hepatology, Kantonsspital St. Gallen, St. Gallen, Switzerland

* [email protected]

Abstract
OPEN ACCESS
Background
Citation: Vögelin M, Biedermann L, Frei P, Vavricka
Thiopurines are known to cause lymphopenia (<1,500 lymphocytes/μl). As severe lympho-
SR, Scharl S, Zeitz J, et al. (2016) The Impact of
Azathioprine-Associated Lymphopenia on the Onset penia (<500C/μl) is associated with opportunistic infections, we investigated severity of thio-
of Opportunistic Infections in Patients with purine-related lymphopenia and development of opportunistic infections in our tertiary
Inflammatory Bowel Disease. PLoS ONE 11(5):
referral centre.
e0155218. doi:10.1371/journal.pone.0155218

Editor: John Green, University Hospital Llandough,

Methods
UNITED KINGDOM
We retrospectively screened medical records of 1,070 IBD patients and identified 100 indi-
Received: February 16, 2016
viduals that developed a total of 161 episodes of lymphopenia during thiopurine treatment
Accepted: April 26, 2016
between 2002 and 2014. Occurrence of opportunistic infections was documented. A control
Published: May 23, 2016 group consisted of IBD patients receiving thiopurines but without developing lymphopenia.
Copyright: © 2016 Vögelin et al. This is an open
access article distributed under the terms of the Results
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any Of a total of 161 episodes of lymphopenia, 23% were severe (<500C/μl). In this subgroup,
medium, provided the original author and source are thiopurine dosing was modified in 64% (dosage reduction: 32%, medication discontinued:
credited. 32%). We identified 9 cases (5.5%) of opportunistic infections, of which only two occurred
Data Availability Statement: All relevant data are during severe lymphopenia. One opportunistic infection (4.5%) was identified in the control
within the paper. group. No association was found between opportunistic infections and severity of lympho-
Funding: This research was supported by research penia. All patients who suffered from opportunistic infections were receiving additional
grants from the Swiss National Science Foundation to immunosuppressive medication.
MS (Grant No. 314730-146204 and Grant No. CRSII3
154488/1) and to GR: the Swiss IBD Cohort Study
(Grant No. 3347CO-108792). Conclusion
Competing Interests: The authors have declared Our patients treated with thiopurines rarely developed severe lymphopenia and opportu-
that no competing interests exist. nistic infections did not occur more often than in the control group. A careful monitoring of

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 1 / 17

 Azathioprine and Opportunistic Infections

lymphocytes and prophylactic adjustment of thiopurine therapy might contribute to this

low incidence.

Introduction
Azathioprine (AZA) is the best known agent of a family known as thiopurines or purine anti-
metabolites. AZA was developed approximately fifty years ago and is well established in the
treatment of inflammatory bowel disease (IBD) up to the present day, together with its metabo-
lite 6-mercaptopurine (6-MP). It is further being used in other chronic inflammatory diseases,
chemotherapy and organ transplant recipients [1–4].
The advantage of AZA/6-MP over placebo for maintenance of remission in Crohn’s disease
(CD) has been proven by several meta-analyses showing good evidence for its steroid sparing
effect [5, 6]. Due to their slow onset of action, they are usually introduced in combination with
fast-acting immunosuppressive drugs, such as steroids that cover the time period until AZA/
6-MP takes full effect. Thus, it is conceivable that a recent Cochrane analysis concludes that
there is no significant benefit of thiopurine monotherapy for the induction of remission in CD
[7–9]. In ulcerative colitis (UC), the available literature displays a similar situation with good
evidence for the efficacy of thiopurines for maintenance therapy [10, 11], but not for induction
of remission [9, 12].
As an inactive prodrug AZA is converted to 6-MP and further, via multi-enzymatic process-
ing, to 6-methyl-mercaptopurine (6-MMP), 6-thiouric acid (6-TUA) and to active metabolites
that can be summarized as 6-thioguanin nucleotides (6-TGN) and 6-methylmercaptopurine
ribonucleotides (6-MMPRs). 6-TGN are primarily responsible for both, the cytotoxic potential
and the immunosuppressive effect of these agents [13–16]. They are structurally similar to
endogenous purine-bases and have a cytotoxic effect on leucocytes by interfering with DNA
synthesis [17]. Furthermore, inhibition of the Ras-related C3 botulinum toxin substrate 1
(Rac1) resulting in T-cell apoptosis is presumed [18]. In addition to 6-TGN, 6-MMPRs are also
considered to have an immunosuppressive effect by interfering with the synthesis of purine-
bases [19, 20]. Eventually, although some details about the mode of action of AZA/6-MP
remain unknown to date, it can be concluded that the immunosuppressive effects of thiopurine
drugs are mediated by an interplay of various thiopurine metabolites with the cellular and
humoral immune system [21–24].
Besides its benefits in the treatment of IBD, AZA is known to potentially cause a broad spec-
trum of adverse effects, reaching from mild (e.g., itching) to severe (e.g., pancreatitis, liver fail-
ure and severe myelosuppression) [22, 25]. Some side effects can be related to specific
metabolites, whereas in other cases the exact pathogenesis remains unclear. The hepatotoxic
potential for example has been found to be predominantly triggered by 6-MMP and 6-MMPR
[16, 26, 27].
In this study, we particularly focus on the development of lymphopenia as a crucial compo-
nent of myelosuppression. Data on the incidence of AZA related lymphopenia are highly vari-
able, depending on the definition and grading of lymphopenia as well as the study setting, with
reported fractions in IBD patients between 10% up to over 30% [28, 29]. In this work, we grad-
uated lymphopenia according to lymphocyte cell counts into mild (1,499–1,000 C/μl), moder-
ate (999–500 C/μl), severe (499–200 C/μl) and most-severe (< 200 C/μl).
Lymphopenia may increase the risk of serious opportunistic infections, as seen in HIV posi-
tive patients, primary immunodeficiency, idiopathic CD4+-Lymphopenia and chronic

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 2 / 17

 Azathioprine and Opportunistic Infections

immunosuppressive therapy [30–35]. It has further repeatedly been associated with infectious
complications in various chronic inflammatory diseases [33, 36, 37]. Also IBD patients bear a
greater risk of opportunistic infections and, according to current literature, AZA/6-MP seem to
be at least partially responsible for this [36]. However, data regarding the incidence of infec-
tious complications in patients with IBD or other chronic inflammatory diseases differ consid-
erably, which can, to some extent, be ascribed to different study settings: Data from the TREAT
registry, a large North American cohort study, reports serious infections to occur at a rate of
1.7% in patients with CD, regardless of their medical treatment [38]. In a pooled safety analysis
of 7 CD and 3 UC cohorts, Lichtenstein et al. counted serious infections in 4.6% of CD and
5.4% of UC (total IBD: 4.9%) patients treated with immunomodulators such as AZA/6-MP or
methotrexate (MTX) [39]. When patients with different chronic inflammatory diseases were
investigated by Glueck et al. in a German cohort, the rate of infections requiring hospitalisation
increased to 13.8% in patients that received AZA/6-MP with or without concomitant steroids
[33]. The latter study is of particular interest, as it proved lymphopenia with cell counts < 600
C/μl, respectively CD4+ < 250 C/μl, to significantly increase the risk for development of
infections.
Considering that the immunosuppressive properties of AZA are mainly achieved by its
cytotoxic effects on T-lymphocytes, it is legitimate to assume that mild to moderate lymphope-
nia should rather be interpreted as a parameter of a sufficient immunosuppression, than solely
as an adverse event.
Therefore, our centre, in accordance with global clinical experience, pursues a policy that
tolerates and even targets mild up to moderate lymphopenia but takes prophylactic measures
when severe lymphopenia (< 500 C/μl) occurs during treatment with thiopurines. Such pro-
phylactic measures usually include dose reduction or cessation of AZA/6-MP as it has been rec-
ommended in earlier studies [25].
In this work, we analysed the occurrence of opportunistic infections in patients with thio-
purine-related lymphopenia at the IBD clinic of the Division for Gastroenterology and Hepa-
tology at the University Hospital Zurich.

Methods
Patient Selection Process
In this retrospective study we conducted a systematic computer based search for the appear-
ance of the terms ‘azathioprine’, ‘6-mercaptopurine’ or ‘6-thioguanine’ in health records of
1,070 patients from the Swiss IBD Cohort, who were being treated at our clinic between
2002–2014. The records of patients with thiopurines were screened for lymphopenia
(defined as < 1,500 lymphocytes/μl) during treatment with this medication. Patients with
insufficient documentation of either lymphocyte counts or medication were excluded from
analysis. All data was obtained from electronically filed medical reports of the IBD outpa-
tient clinic at the Division of Gastroenterology and Hepatology at the University Hospital
Zurich, Switzerland.

Demographic and Clinical Characteristics

We collected data with regards to patients’ disease and treatment characteristics as well as
occurrence of opportunistic infections. Diagnosis of IBD had always been verified by endo-
scopic and histological examinations. The physician collecting and analysing the data was not
involved in patient care. We recorded sex, age, smoking habits and type of disease (CD or UC)
including date of first diagnosis. Whenever possible we obtained information about disease
characteristics such as the existence of fistulas and/or abscesses, as well as extraintestinal

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 3 / 17

 Azathioprine and Opportunistic Infections

manifestations. In the case of CD, the Vienna Classification was documented. We further
noted the course of treatment with purine analogues including date of initiation and cessation,
together with dosage and dose-reductions when lymphopenia appeared. Co-medication for
IBD (e.g., topical or systemic steroids, 5-aminosalicylic acid containing agents (5-ASA), tumor
necrosis factor α inhibitors (TNFα-Inhibitors), methotrexate (MTX), cyclosporine A (CsA),
and tacrolimus) was registered separately. Lymphopenia was defined as complete lymphocyte
count < 1,500 cells/μl (C/μl) and lowest lymphocyte counts (referred to as nadir) during treat-
ment with AZA/6-MP were graded in 4 groups (mild: 1,499–1,000 C/μl, moderate: 999–500
C/μl, severe: 499–200 C/μl, most-severe: < 200 C/μl). The beginning, duration and recovery of
lymphopenia to standard values (> 1,500 C/μl) were assessed chronographically.
We searched for occurrence of opportunistic infections during AZA/6-MP-associated lym-
phopenia, registered the pathogen, site of infection and its outcome regarding survival. Oppor-
tunistic infections were defined as those infections that occur more frequently or solely in
patients with a compromised immune system, as e.g. AIDS-defining illnesses [40]. Oral, peria-
nal or vaginal candidiasis was diagnosed by the characteristic clinical appearance and response
to therapy with antimycotics. In case of oesophageal candidiasis, endoscopic and histological
confirmation was obtained. Diagnosis of CMV colitis was confirmed by either positive CMV
IgM, IgG and elevated viral load (CMV DNA >15,000 copies/ml) or by positive PCR for CMV
DNA and immunohistochemical detection of CMV infected cells in biopsy specimens.
The same procedure, except for the lymphocyte counts, was applied to the control group
which consisted of 22 IBD patients who also received AZA, 6-MP or 6-TG but did not develop
lymphopenia. The small sample size of this group was mainly due to inconsistent laboratory
records in this retrospective search.
All patients in this study are taking part in the prospective multicentre Swiss IBD Cohort
Study and gave written informed consent. The entire data set was anonymized. The study was
approved by the local Ethical Committee of the University of Zurich (EK-1316)

Statistical Analysis
The IBM Statistical Package for the Social Sciences (SPSS, Chicago, IL; version 22) was used to
analyse both groups with descriptive statistics. Non-parametric data was expressed as absolute
numbers and percentages. Numeric variables were presented as the mean plus standard devia-
tion. A 95% confidence interval and the median with interquartile range were calculated for
lymphocyte nadirs. To visualize the distribution of the data we used histograms, boxplots and
bar charts. To achieve better readability, percentages displayed in tables were rounded.

Results
Demographic Aspects and Disease Characteristics
Out of all 1,070 patients in our IBD clinic seen during 2002–2014, 385 patients had AZA or
6-MP mentioned in their medical record at any time (Fig 1). Out of those 385, 263 patients had
to be excluded from analysis due to insufficient documentation of either lymphocyte counts or
medication. We included 100 patients with IBD (56 with CD, 44 with UC; 48 females) and lym-
phopenia under AZA/6-MP (71% > 500 C/μl, 29% < 500 C/μl). The control group consisted
of the remaining 22 patients, who showed standard lymphocyte values (> 1,500 C/μl) through-
out their medical documentation.
Demographic aspects and clinical characteristics are summarized in Table 1. Extraintestinal
manifestations were present in 34 (61%) of CD and in 14 (32%) of UC patients. In two (3.6%)
CD patients this information remained unknown. With 25 (44.6%) cases, almost half of the
patients with CD suffered from fistulae or abscesses. Smoking habits could only be evaluated in

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 Azathioprine and Opportunistic Infections

Fig 1. Selection process. This figure shows the patient selection process.
doi:10.1371/journal.pone.0155218.g001

72 (72%) of all subjects. Interestingly, 13 (23.2%) CD patients but only 6 (13.6%) UC patients
were smokers. In both groups, 10 (CD: 17.9%, UC: 22.7%) persons quit smoking sometime
before development of lymphopenia and 10 (CD, 17.9%), respectively 16 (UC, 36.4%), were
non-smokers.

Table 1. Patient and disease characteristics.

Patients with lymphopenia Patients without lymphopenia (control

group)

Total CD UC Total CD UC
Number 100 100% 56 56% 44 44% 22 100% 17 77% 5 23%
Female 48 48% 27 48% 21 48% 11 50% 8 47% 3 60%
Male 52 52% 29 52% 23 52% 11 50% 9 53% 2 40%
Average age at IBD diagnosis (y) 28 SD: ± 10.68; range: 12.52–56.68 27 30 24 24 24
Extraintest. Manifestations 48 48% 34 61% 14 32% 15 68% 10 59% 5 100%
Unknown 2 2% 2 34% 0 0% 0 0% 0 0% 0 0%
Abscess/fistula 25 25% 25 45% 0 0% 6 27% 6 35% 0 0%
Unknown 2 2% 2 4% 0 0% 0 0% 0 0% 0 0%
Smoking habits:
Smoker 19 19% 13 23% 6 14% 3 14% 3 18% 0 0%
Former smoker 20 20% 10 18% 10 23% 3 14% 2 12% 1 20%
Non-smoker 33 33% 10 18% 16 36% 7 32% 4 24% 3 60%
Unknown 28 28% 16 29% 12 27% 9 41% 8 47% 1 20%

Patient and disease characteristics of the lymphopenia and the control group

doi:10.1371/journal.pone.0155218.t001

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 5 / 17

 Azathioprine and Opportunistic Infections

Table 2. Medication.

Total CD UC Total episodes CD UC

patients
Number 100 100% 56 56% 44 44% 161 100% 85 53% 76 47%
AZA 81 80% 43 77% 38 86% 135 84% 65 76% 70 92%
6-MP 16 16% 10 18% 6 14% 21 13% 15 18% 6 8%
6-TG 3 3% 3 5% 0 0% 5 3% 5 6% 0 0%
Average dosage (mg/d):
AZA 138 131 145 135 SD: ±47.63; range: 50–250 135 135
6-MP 73 83 58 73 SD: ±22.23; range: 25–100 78 58
6-TG 30 30 0 26 SD: ±8.94; range: 20–40 26 0
Stopped during lymphopenia 27 27% 19 34% 8 18% 31 19% 20 24% 11 14%
Dose reduction 18 18% 9 16% 9 20% 28 17% 15 18% 13 17%
Co-medication:
Anti-TNFalpha 16 16% 13 23% 3 7% 25 16% 20 24% 5 7%
Steroids systemic 57 57% 25 45% 32 73% 76 47% 30 35% 46 61%
Steroids topical 28 28% 15 27% 13 30% 42 26% 19 22% 23 30%
5-ASA 46 46% 10 18% 36 82% 72 45% 14 16% 58 76%
MTX/CsA/Tacrolimus 6 6% 2 4% 4 9% 9 6% 2 2% 7 9%
No co-medication 13 13% 12 21% 1 2% 28 17% 24 28% 4 5%

Medication during lymphopenia displayed separately for all patients and all episodes of lymphopenia.

doi:10.1371/journal.pone.0155218.t002

Medication
AZA was the most commonly used purine analogue (n = 81; 81%), followed by 6-MP (n = 16;
16%) and 6-TG (n = 3; 3%). At nadir of lymphopenia (lowest lymphocyte count), most patients
were receiving additional medication as part of their IBD-treatment. 13 (13%) patients were on
sole treatment with purine analogues. In CD, systemic steroids were the most frequently used
co-medication whereas 5-ASA was the preferred agent in UC. The average dosage of each type
of purine analogue was 138 mg/d (SD: ± 43.14) for AZA, 73 mg/d (SD: ± 19.30) for 6-MP and
30 mg/d (SD: ± 10.00) for 6-TG, respectively.
Thiopurine therapy was stopped in 31 (19%) cases of lymphopenic episodes overall and
dosage was reduced during lymphopenia in 28 (17%) patients. When subdivided according to
severity of lymphopenia, these numbers become more revealing: Regarding severe lymphope-
nia (< 500 C/μl), thiopurine was altered in 24 (64%) individuals (dosage reduction: 32%, medi-
cation discontinued: 32%). This is more than twice as often as in the group with lymphocytes
between 500–1,500 C/μl, in which therapy was reduced in only 16 (13%) and stopped in 19
(15%) patients. Further details regarding medication are displayed in Table 2.

Lymphopenia
In the group of 100 patients, 161 episodes of lymphopenia occurred (53% (n = 85) patients
with CD, 47% (n = 76) patients with UC). 32% (n = 32) had fluctuating lymphocyte counts and
experienced more than one episode of lymphopenia (range: 2–7 episodes). 3 episodes recurred
when dose was increased again after a previous reduction and another 3 after foregoing cessa-
tion of thiopurine therapy. In 6 cases lymphopenia reappeared when dose was directly
increased without having been reduced before. 8 episodes recurred despite dose reduction and
no adjustment in thiopurine therapy was made prior to the remaining 42 episodes. The

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 Azathioprine and Opportunistic Infections

lymphocyte count remained > 500 C/μl in the majority of all lymphopenic episodes (n = 124;
77%). The nadir was between 200–499 C/μl in 18.63% (n = 30). Most severe lymphopenia
(< 200 C/μl) occurred only rarely, accounting for 4.35% (n = 7). The two subgroups CD and
UC showed a similar pattern of distribution over all four subgroups of lymphopenia levels
(1,000–1,499 C/μl, 500–999 C/μl, 200–499 C/μl and < 200 C/μl).
Mild to moderate lymphopenia (1,499–500 C/μl) recovered to standard values (> 1,500
C/μl) more often as compared to those with cell counts < 500 C/μl (100 (81%) mild to moder-
ate vs. 16 (32%) severe to most-severe). 73% (n = 37) of mild to moderate lymphopenia recov-
ered spontaneously, but only 44% (n = 7) of those with severe to most-severe lymphopenia.
Conversely, patients with lymphocyte counts < 500 C/μl recovered more frequently after thio-
purine dose reduction (10 (10%) mild to moderate vs. 4 (25%) severe to most-severe) and dis-
continuation of thiopurine therapy (17 (17%) mild to moderate vs. 5 (31%) severe to most-
severe). The remaining 28% (n = 45) had not reached standard values again by the latest docu-
mented blood examination at the University Hospital Zurich. The exact numbers and further
details about the chronological course of lymphopenia are summarized in Table 3.

Opportunistic Infections
Nine patients (5 females; 5 CD, 4 UC) developed an opportunistic infection during lymphope-
nia. Three patients suffered from CMV colitis and in 4 cases oral and/or oesophageal candidia-
sis was diagnosed. One patient developed vaginal and perianal candidiasis and one other
person had to be treated for herpes zoster (Table 4).
The opportunistic infections occurred during lymphopenic episodes with an average nadir
of 486 C/μl in CD and 1,120 C/μl in UC. Table 5 shows a broad distribution of lowest lympho-
cyte counts during opportunistic infections with a wide interquartile range (755 C/μl). Median
and mean lymphocyte nadirs lay more than 200 C/μl from each other. Only three out of nine
cases did not reach regular lymphocyte counts again by the day of our inquiry or by the end of
observation in our hospital. Opportunistic infections were not significantly more frequent
(6.67%, n = 2 out of 30 episodes) in the subgroup with nadirs between 200–499 C/μl than in
the one with 1,000–1,499 C/μl (4.76%, n = 3 out of 63 episodes). Opportunistic infections were
not observed in the patient group with severe lymphopenia (< 200 C/μl). Fig 2 gives a brief
overview of this distribution.
Regarding treatment with purine analogues, 7 out of 9 patients received AZA. The mean
AZA dosage (171 mg/d) was almost 40 mg higher than the average AZA dosage in all patients
with lymphopenia (135 mg/d). One patient each received 6-MP (75 mg/d) and 6-TGN (20 mg/
d). AZA/6-MP was reduced or discontinued in only 4 individuals. In two cases this was due to
very low lymphocyte counts < 500 C/μl, in one because of CMV colitis and in another one
AZA was considered ineffective. In the remaining 5 cases, therapy with purine analogues was
continued despite the existence of an opportunistic infection. The average lymphopenic period
lasted 428 days (range: 29–907 days) with dosage reduction and 335 days (range: 6–1,072 days)
when thiopurines were stopped. Four out of the remaining 5 patients in whom AZA was con-
tinued recovered to standard values after a mean of 206 days (range: 3–1,335 days) (Table 6).
6-TGN and 6-MMP levels were measured in 3 out of 9 cases of opportunistic infections. Two
individuals had 6-TGN levels within the therapeutic margin (768 and 765 pmol/8 108 Erythro-
cytes), one showed an insufficient level with 196 pmol/8 108 Erythrocytes. No patient with
opportunistic infection had a 6-MMP level above 5,000 pmol/8 108 Erythrocytes.
All patients who suffered from an opportunistic infection received other medication addi-
tionally to AZA/6-MP/6-TGN. Being treated adequately, all subjects fully recovered from their
infection.

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 7 / 17

 Table 3. Lymphopenia.
Total episodes of CD UC Thiopurine dose Thiopurine Recovery*: Spontaneously upon thiopurine upon thiopurine
lymphopenia reduction cessation dose reduction cessation

Number 161 100% 85 53% 76 47% 28 17% 31 19% 116 72% 80 50% 14 9% 22 14%
1'000–1'499 C/μl 63 39% 33 39% 30 39% 6 10% 5 8% 53 84% 44 83% 4 8% 5 9%
500–999 C/μl 61 38% 31 36% 30 39% 10 16% 14 23% 47 77% 29 62% 6 13% 12 26%
200–499 C/μl 30 19% 17 20% 13 17% 12 40% 7 23% 15 50% 7 47% 4 27% 4 27%
< 200 C/μl 7 4% 4 5% 3 4% 0 0% 5 71% 1 14% 0 0% 0 0% 1 100%
> 500 C/μl 124 77% 16 13% 19 15% 100 81% 73 73% 10 10% 17 17%
< 500 C/μl 37 23% 12 32% 12 32% 16 32% 7 44% 4 25% 5 31%

Severity and course of lymphopenia.

*Recovery = recover to lymphocyte counts >1,499 C/μl by October 2014 or up to the latest documented blood examination at the University Hospital Zurich
doi:10.1371/journal.pone.0155218.t003

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Azathioprine and Opportunistic Infections
 Azathioprine and Opportunistic Infections

Table 4. Site of opportunistic infection.

Patients with lymphopenia (n = 100)

Organism Site of infection n
Candida oesophagus 3
oral 2
perianal/vaginal 1
Cytomegalovirus (CMV) colon 3
Varicella zoster virus (HHV—3) skin 1
Patients without lymphopenia (n = 22) (control group)
Organism Site of infection n
Cytomegalovirus (CMV) colon 1

Site of infection and pathogen in case of opportunistic infections.

doi:10.1371/journal.pone.0155218.t004

Control Group of Patients without Lymphopenia

The control group consisted of 22 patients (11 females, 17 (77%) CD). 6 individuals from the
CD subgroup (35%) suffered from an abscessing/fistulising course of their disease, but no one
in the UC subgroup did. As regards to use of tobacco among our CD control patients, 3 (18%)
declared to be smokers, 2 (12%) were former smokers, 4 (24%) non-smokers and in 8 cases
(47%) smoking habits could not be evaluated. No current smokers were detected in the UC

Table 5. Lymphopenia during opportunistic infections.

Opportunistic infections Total episodes of lymphopenia

IBD (n = 161) CD (n = 85) UC (n = 76)

Total 9 6% 5 6% 4 5% 161
1'000–1'499 C/μl 3 5% 63
500–999 C/μl 4 7% 61
200–499 C/μl 2 7% 30
< 200 C/μl 0 0% 7
Mean duration of lymphopenia (d) 117 257
Nadir during opportunistic infection (n = 9) [C/ul]
Mean 767 486 1120
Std. Error of Mean 145
Median 550
Std. Deviation 436
Range 1210
Minimum 250
Maximum 1460
25th percentile 480
50th percentile 550
75th percentile 1235
Interquartile Range 755

Occurrence of opportunistic infections per lymphopenia subgroup, details about lowest lymphocyte counts (nadir) during opportunistic infection and mean
duration of lymphopenia.

doi:10.1371/journal.pone.0155218.t005

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 Azathioprine and Opportunistic Infections

Fig 2. Severity of lymphopenia. Thiopurine-associated episodes of lymphopenia and opportunistic infections, subdivided
according to severity of lymphopenia.
doi:10.1371/journal.pone.0155218.g002

group. Here, 3 patients (60%) were non-smokers, one person (20%) stopped smoking and one
remained unknown. Patients’ and disease characteristics are summarized in Table 1.
Similar to the lymphopenia group, the majority (86%, n = 19) of patients in the control
group also received AZA as a purine analogue (mean dosage 175 mg/d, higher dosage in the
UC than in the CD subgroup). 14% (n = 3) were treated with 6-MP (average dose 67 mg/d)
and no one received 6-TGN. The mean duration of therapy with AZA/6-MP was 804 days
(Table 7).
Of all 22 cases, one (5%) developed an opportunistic infection: One male UC patient suf-
fered from CMV colitis while being treated with AZA 200 mg/d for 639 days. During the infec-
tion he received co-medication with topical and systemic steroids and a 5-ASA preparation.

Discussion
In this retrospective search we investigated the occurrence of thiopurine related opportunistic
infections in IBD patients and found no association between opportunistic infections and
severity of lymphopenia, while prophylactic adjustment of medication might have contributed
to this result.
In our study, opportunistic infections were of mostly mild character (e.g. muco-cutaneous
candidiasis, herpes zoster) and occurred in only 5.6% of all patients with lymphocyte
counts < 1,500 C/μl. When calculated for severe lymphopenia only (< 500 C/μl) the frequency
even dropped to 1.24%, which is astonishingly low, compared to the studies mentioned above
[33, 39]. The incidence further decreased when CMV colitis, an infection that is debatable to be
counted as an opportunistic infection, was excluded. In our cohort, CMV colitis occurred
equally often in patients with lymphopenia as in the control group. To date, to the best of our
knowledge, no precise data regarding overall prevalence of CMV reactivation in IBD exist, to
which we could compare our patient collective. Most of the reports that exist on this topic
included only a narrowed patient group or used different diagnostic criteria [41].
Lymphopenia has mainly been attributed to exceeding 6-TGN levels [42–44]. These metab-
olites accumulate in the tissue and can be measured in erythrocytes, but its widespread use for
drug monitoring is currently a point at issue and needs to be further evaluated [9, 45]. The

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 10 / 17

 Azathioprine and Opportunistic Infections

Table 6. Details of opportunistic infections during lymphopenia.

#1 #2 #3 #4 #5 #6 #7 #8 #9 Summary /
Mean
Sex (W = 1, M = 2) 1 1 2 2 1 1 2 1 2 5 W, 4 M
IBD (CD = 1, 1 2 1 1 1 1 2 2 2 5 CD, 4 UC
UC = 2)
Age at first 45 34 16 48 34 22 14 56 30 33
diagnosis (y)
Extraint. 1 2 1 1 1 2 2 2 4
manifestations
(1 = yes, 2 = no)
Abscess/fistula 2 2 2 2 1 2 2 2 1
(1 = yes, 2 = no)
smoker (1 = yes, 2 3 3 2 3 3 0 S, 2 NS, 4
2 = no, 3 = former) ExS, 3
unknown
Dosage AZA (mg/ 175 150 150 150 200 175 200 171
d)
Dosage 6-MP (mg/ 75 75
d)
Dosage 6-TGN 20 20
(mg/d)
AZA/6-MP stop/ 2 3 1 2 3 2 1 2 2 2 stopped, 2
reduced DURING reduced, 5
lymphopenia no alteration
(1 = stopped,
2 = no alteration,
3 = reduced)
Reason for lymphopenia paused lymphopenia no
reduction/stop because CMV remission
colitis achieved
Co-medication Anti-TNFa, steroids 5-ASA steroids local, steroids steroids 5-ASA steroids steroids
during nadir MTX/ system. & MTX/ system., system. system. & system.,
Tacrolimus/ local, 5-ASA Tacrolimus/ 5-ASA local, 5-ASA 5-ASA
CsA CsA
Nadir during 430 550 540 680 250 530 1410 1060 1460 768
treatment with
purine analogue (C/
ul)
Duration: Start 4807 86 174 180 44 433 452 192 796
purine analogue—
nadir (d)
Lymphocytes 2 2 1 1 2 1 1 1 1 6 yes, 3 no
recovery (1 = yes,
2 = no)
Duration: stop 4 19 12
purine analogue—
reaching standard
values (d)
Duration: nadir— 6 164 55 20 92 7 57
reaching standard
values (d)
Duration 6 256 320 20 92 7 117
lymphopenia (d)
Diagnosis Oesophageal Oesophageal CMV colitis Oesophageal Oral Herpes CMV Vaginal and CMV
opportunistic candidiasis candidiasis and and oral candidiasis zoster colitis perianal colitis
infection concomitant candidiasis candidiasis
hepatitis

Detailed patient and disease characteristics for lymphopenia-associated opportunistic infections.

doi:10.1371/journal.pone.0155218.t006

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 11 / 17

 Azathioprine and Opportunistic Infections

Table 7. Control group.

Total CD UC
Number 22 100% 17 77% 5 23%
Medication
AZA 19 86% 15 88% 4 80%
6-MP 3 14% 2 12% 1 20%
6-TGN 0 0% 0 0% 0 0%
Average dose (mg/d):
AZA 139 SD: ±35.59; range: 50–200 129 175
6-MP 67 SD: ±14.43; range: 50–75 63 75
6-TGN 0 SD: ±8.94; range: 20–40 0 0
Unknown 1 1 0
Mean duration of therapy with purine analogues (d) 804 803 810
Opportunistic infection: 1 5% 0 0% 1 20%
Dosage AZA (mg/d) 200
Co-medication during opport. Infection steroids syst. and local, 5-ASA
Diagnosis opport. infection CMV colitis, 10.02.2012

Details about thiopurine therapy and occurrence of opportunistic infections in the control group.

doi:10.1371/journal.pone.0155218.t007

intracellular concentration depends on many factors, such as dosage and inter-individually

highly variable bioavailability of AZA/6-MP, enzyme activity of the thiopurine-methyltransfer-
ase (TPMT) and interactions with other drugs. Levels exceeding the therapeutic margin can
lead to severe myelosuppression with potentially lethal septic complications [46–52]. As
6-TGN is also considered to primarily mediate the immunosuppressive effects of AZA, a nar-
row therapeutic margin has to be presumed. Therefore the pharmacodynamics of AZA suggest
that mild lymphopenia might be proof of a sufficient immunosuppression with therapeutic
intracellular concentration of 6-TGN, whereas severe lymphopenia and its consequences result
from excessive levels of the latter [53]. It is consequently of crucial importance to differentiate
between mild to moderate (1,499–500 C/μl) and severe lymphopenia (<500 C/μl) when inter-
preting data about lymphopenia.
As in our data, opportunistic infections were not more frequent in case of severe lymphope-
nia. One possible interpretation is that thiopurine induced lymphopenia might not be as
immune compromising as lymphopenia in other medical conditions, such as e.g. advanced
HIV infection. However, we believe that prophylactic therapy adjustment, as is was often made
in our collective with severe lymphopenia, could have substantially contributed to such a low
incidence of opportunistic infections in this subgroup. It may also be a reason for the compara-
tively few patients with cell counts < 200 C/μl, as a threshold of 500 C/μl for prophylactic dos-
age adjustment might have stopped a further decline. Additionally, other authors have
described severe lymphopenia to be associated with infectious complications. Glueck et al. for
example identified in a German cohort total lymphocyte counts < 600 C/μl and CD4+ lympho-
cyte counts < 250 C/μl as individual risk factors for the development of infections requiring
hospitalisation (not limited to opportunistic infections) in patients with chronic inflammatory
diseases, irrespective of their immunosuppressive therapy [33].
Regarding therapy adjustments, it is not surprising that both reduction and cessation were
made more frequently in patients with severe to most-severe lymphopenia. However, as no
official recommendation for the adjustment of thiopurine therapy exists, no adaption of dosage
was made in a significant amount of the cases with severe lymphopenia. The decision whether

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 12 / 17

 Azathioprine and Opportunistic Infections

to reduce thiopurine dosage or not is left to the treating physician’s discretion and dependent
not only on the absolute lymphocyte count, but also on diseases severity, course of disease, co-
medication and many other factors. The adjustments made in the collective with only mild to
moderate lymphopenia may have mainly been due to reasons other than lymphopenia.
It needs to be considered that lymphocyte counts are also influenced by many other vari-
ables such as physical or psychological stress, co-medication, disease activity and concomitant
diseases. Especially viral infections can considerably influence lymphocyte counts and suscepti-
bility to infections. Additionally, aging related attenuation of immune-competence is of impor-
tance [54–58].
Particular emphasis has to be laid on co-medication, as all patients with infections during
lymphopenia received additional immunosuppressive medication. Combined immunosup-
pression is likely to bear a greater risk not only for development of lymphopenia [28] but also
for opportunistic infections [33, 36]. For CMV re-activations specifically, an increased risk for
active infection in patients with double immunosuppression consisting of AZA and TNFα-
inhibitors was described [59]. Furthermore, systemic or local steroids bear a risk for develop-
ment of mucocutaneous candidiasis [36]. Systemic steroids were the most frequent co-medica-
tion among patients with opportunistic infections (as well as in the whole study population)
and could explain the high number of mucocutaneous candidiasis in this group. Thus, our
findings support the growing evidence that a combined immunosuppression in patients with
IBD increases the risk for opportunistic infections.
Eventually, the difficulty for the physician in case of lymphopenia during thiopurine treat-
ment is to adapt therapy in a way that achieves a remission maintaining immunosuppressive
level but does not drastically increase the risk for infectious complications due to myelosup-
pression. Therefore, a regular blood count monitoring is of major importance and cannot be
replaced by other prophylactic measurements like TPMT genotyping or metabolite monitoring
[9, 60, 61].
Our study has weaknesses and strengths. A strength is the fact that health records for IBD
outpatient and inpatient consultations at the University Hospital of Zurich are standardized,
which results in a homogenous data quality. Furthermore, our study analysed a cohort which is
representative for a characteristic Western IBD patient collective. The fact that it was con-
ducted at a tertiary university hospital can be interpreted as an advantage as well as a disadvan-
tage. As difficult cases such as opportunistic infections in IBD patients tend to converge in
tertiary centres, the probability was high that such cases would have been registered in our cen-
tre. This on the other hand could reflect a selection bias that overestimates the incidence of
opportunistic infections among patients with lymphopenia. Accordingly, the low rate of severe
infection even in sever lymphopenia appears somewhat reassuring.
Several methodological limitations of the data presented need to be considered. The retro-
spective study design substantially limited the number of patients that could be included: Labo-
ratory results for lymphocyte counts were partially incomplete, due to intermittent observation
at our centre. This could result in an overestimated duration of lymphopenic episodes (date of
first value < 1,500 C/μl—date of recovery to a value > 1,500 C/μl). Serum levels of 6-TGN and
6-MMP in particular, were only recorded inconsistently and could therefore not be correlated
with lymphopenia or opportunistic infections. IBD patients in remission, with mild flares or
mild opportunistic infections such as oral thrush are often managed by district hospitals, by
gastroenterologists in private practices or by general practitioners. The consecutive small sam-
ple size of our control group did not allow us to match for sex and age or to apply comparable
statistics. A prospective study design would enable to measure T-cell subsets instead of total
lymphocyte counts, as these were not determined in standard laboratory monitoring. Further-
more, studies conducted at tertiary centres entail a selection bias for patients with active flairs

PLOS ONE | DOI:10.1371/journal.pone.0155218 May 23, 2016 13 / 17

 Azathioprine and Opportunistic Infections

and more severe disease. The high percentage of patients treated with steroids in our collective
could be a reflection of this.
The sheer variety of influencing factors in IBD patients makes a bias-free evaluation of risk
factors extremely complex and costly. Still, given the clinical relevance, it would be interesting
to look at some of them in more detail: As Glueck et al. identified CD4+ lymphocyte counts to
be a better predictor for infectious complications than total lymphocyte counts [33], evaluating
the influence of AZA on the distribution of T-lymphocyte subtypes in future studieswould
enable direct comparison with AIDS patients.
Another point of interest is that an increasing incidence of Pneumocystis carinii pneumonia
(PCP) in IBD has been reported and combined immunosuppressive therapy or lymphocyte
counts < 400 C/μl or <300 C/μl have been suggested as risk factors [62, 63]. Even though anti-
microbial PCP prophylaxis has been proven to be highly effective in HIV negative immuno-
compromised patients [64], there is still no clear evidence for PCP prophylaxis in IBD [62].
In summary and having regard to recent studies, persistent severe lymphopenia (lympho-
cyte count < 500 C/μl) might expose the patient to a greater risk for infectious complications.
Close lymphocyte monitoring and prophylactic dose adjustments in case of thiopurine-
induced severe lymphopenia may be recommended, as this procedure is likely to reduce the
number of infectious complications in these patients. However, when this careful monitoring
and consequent dose adjustment is performed, the risk for infectious complications might be
substantially lowered, maybe even to the risk of those patients not receiving thiopurine therapy.
Further prospective studies are needed to confirm our assumption and to define an exact cut-
off lymphocyte value.

Author Contributions
Conceived and designed the experiments: GR MS. Performed the experiments: MV LB PF SRV
SS JZ MCS MF GR MS. Analyzed the data: MV GR MS. Wrote the paper: MV LB PF SRV SS
JZ MCS MF GR MS.

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