Visceral Leishmaniasis and Immunocompromise as a Risk Factor for the Development of Visceral Leishmaniasis: A Changing Pattern at The Hospital for Tropical Diseases, London

Abstract

A retrospective study of imported VL to the HTD, London including patients diagnosed and/or managed at the HTD between January 1995 and July 2013. We analyse patient demographics, risk factors for developing VL, diagnosis, investigation, management and outcome. Twenty-eight patients were treated for VL at the HTD over an 18 year period. The median age at VL diagnosis was 44 years (range 4-87 years) with a male to female ratio of 2:1. Most patients were British and acquired their infection in the Mediterranean basin. The median time from first symptom to diagnosis was six months with a range of 1-12 months and diagnosis included microscopic visualisation of leishmania amastigotes, positive serological tests (DAT and k39 antibody) or identification of leishmania DNA. Nineteen patients had some form of immunocompromise and this has increased proportionally compared to previously described data. Within the immunocompromised group, the ratio of those with autoimmune disease has increased. Immunocompromised patients had lower cure and higher relapse rates.
The rise of VL in patients with immunocompromise secondary to autoimmune disease on immunomodulatory drugs presents new diagnostic and therapeutic challenges. VL should be a differential diagnosis in immunocompromised patients with pyrexia of unknown origin returning from travel in leishmania endemic areas.

Full text

RESEARCH ARTICLE
Visceral Leishmaniasis and
Immunocompromise as a Risk Factor for the
Development of Visceral Leishmaniasis: A
Changing Pattern at The Hospital for Tropical
Diseases, London
Kate Fletcher
1☯
*, Rita Issa
1☯
, D. N. J. Lockwood
1,2☯
1Hospital for Tropical Diseases and London School of Hygiene & Tropical Medicine, London, United
Kingdom, 2The Hospital for Tropical Diseases, University college Foundation Trust, London, WC1E 6AU,
United Kingdom
☯These authors contributed equally to this work.
*hs08kmf@doctors.org.uk
Abstract
Visceral leishmaniasis (VL) is a parasitic protozoon infection caused by the Leishmania spe-
cies and transmitted by sandflies. Patients acquire VL in five main tropical areas and the
Mediterranean basin, and clinicians from non-endemic regions regularly see infected pa-
tients. We describe the population presenting with VL to the Hospital for Tropical Diseases
(HTD), London and identify risk factors for developing VL.
Methods and Principal Findings
A retrospective study of imported VL to the HTD, London including patients diagnosed and/
or managed at the HTD between January 1995 and July 2013. We analyse patient demo-
graphics, risk factors for developing VL, diagnosis, investigation, management and out-
come. Twenty-eight patients were treated for VL at the HTD over an 18 year period. The
median age at VL diagnosis was 44 years (range 4–87 years) with a male to female ratio of
2:1. Most patients were British and acquired their infection in the Mediterranean basin. The
median time from first symptom to diagnosis was six months with a range of 1–12 months
and diagnosis included microscopic visualisation of leishmania amastigotes, positive sero-
logical tests (DAT and k39 antibody) or identification of leishmania DNA. Nineteen patients
had some form of immunocompromise and this has increased proportionally compared to
previously described data. Within the immunocompromised group, the ratio of those with
autoimmune disease has increased. Immunocompromised patients had lower cure and
higher relapse rates.
Conclusions
The rise of VL in patients with immunocompromise secondary to autoimmune disease on
immunomodulatory drugs presents new diagnostic and therapeutic challenges. VL should
PLOS ONE | DOI:10.1371/journal.pone.0121418 April 1, 2015 1/9
OPEN ACCESS
Citation: Fletcher K, Issa R, Lockwood DNJ (2015)
Visceral Leishmaniasis and Immunocompromise as a
Risk Factor for the Development of Visceral
Leishmaniasis: A Changing Pattern at The Hospital
for Tropical Diseases, London. PLoS ONE 10(4):
e0121418. doi:10.1371/journal.pone.0121418
Academic Editor: Henk D. F. H. Schallig, Royal
Tropical Institute, NETHERLANDS
Received: October 7, 2014
Accepted: February 1, 2015
Published: April 1, 2015
Copyright: © 2015 Fletcher et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This study was supported by the Special
Trustees of the Hospital for Tropical Diseases.
Margaret Armstrong is supported by The Special
Trustees of the Hospital for Tropical Diseases. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.

be a differential diagnosis in immunocompromised patients with pyrexia of unknown origin
returning from travel in leishmania endemic areas.
Introduction
Visceral leishmaniasis (VL) is a parasitic infection caused by the Leishmania species and is
transmitted by the sandfly. [1] The annual global incidence of VL is over half a million cases in
the endemic zones of Nepal, India, Bangladesh, Sudan, Brazil and the Mediterranean basin [2]
With increasing global travel, clinicians from non-endemic regions are encountering more pa-
tients with VL infection.[3,4]
Patients with VL present with chronic pyrexia, anorexia, splenomegaly and pancytopenia.
However there is a spectrum of clinical disease that depends upon the interplay between the
host immune response and the parasite species and load. [5,6]
In healthy, immunocompetent hosts, leishmania protozoa are killed by T- helper cells (Th),
especially Th-1. Th-1 secrete several cytokines (IL-2, INF gamma and TNF alpha) recruiting
and activating macrophages that phagocytose the cells with leishmania amastigotes.[7] In the
immunosuppressed patients, T cell responses are inadequate and patients have increased sus-
ceptibility to developing clinical disease, experience a more severe disease course and have
higher rates of relapse. [4,8] HIV infection is an established risk factor for developing VL.
[9,10]
In the UK patients with VL are treated at the HTD, London. Whilst risk factors for develop-
ing VL in endemic zones are well defined, [11] there is little published evidence of the risk fac-
tors for developing VL in a non-endemic setting.
We present a retrospective study of imported VL cases diagnosed and/or managed at the
HTD, London between 1995 and 2013, and identify new risk factors for developing VL.
Materials and Methods
Demographic and basic clinical details for all patients seen at the HTD are prospectively col-
lected onto a database (Microsoft Access). Twenty-eight patients with VL were identified and
the case notes and computerized records of these patients were reviewed. Using a standardised
electronic proforma we recorded demographic data and information on past medical history,
time to and method of diagnosis, treatment and outcomes.
The study was reviewed and approved by the Audit and Research Committee at the Hospital
for Tropical Diseases, London who granted ethical approval for the study and stated that indi-
vidual patient consent was not required as this was a retrospective case note review where the
data was fully anonymised and de-identified prior to analysis.
Case definitions
Visceral Leishmaniasis. Symptoms and signs suggestive of VL (chronic pyrexia, spleno-
megaly, pancytopenia) AND laboratory diagnosis of VL as defined below:
1. Visualisation of amastigotes in aspirated tissue material or biopsied tissue sections.[12]
2. A positive Direct Agglutination Test (DAT) which detects antibodies to leishmania proto-
zoa.[12]
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3. A positive rK39 rapid antibody test which detects antibodies to a protein-encoding gene
(K39) found in leishmania species.[13,14]
4. Identification of Leishmania DNA using polymerase chain reaction techniques [12]
Immunocompromise. The authors discussed each patient and agreed that one or more of
the domains below were satisfied. Four categories of immunocompromise were created and
each patient was reviewed to see if they belonged to any category.
1. Condition known to cause immunocompromise
2. Treatment with immunosuppressive drugs
3. Immunocompromising co-morbidity
4. Lifestyle known to cause immunocompromise
Cure. Completion of treatment course AND resolution of clinical signs and symptoms
AND improvement or normalisation of laboratory parameters
Relapse. Achieved cure as defined above AND development of new episode of VL as
defined above
Results
There were 28 patients with VL, the male to female ratio was 2:1 and median age at diagnosis
44 years. Two thirds were British in origin, with the remainder from Algeria, Cyprus, Eritrea,
Ethiopia, India, Italy and Spain.
Patients holidayed in an endemic region (16 patients). Three patients lived in endemic
zones and subsequently emigrated, two patients were visiting friends and relatives (VFR) and
two patients were working. See below for (Fig 1) more detail on areas of acquisition of VL.
Nine patients were identified as immunocompetent and nineteen as immunocompromised.
The data for these two categories will be presented separately.
Immunocompetent Patients
Median age at diagnosis was 38 and the male to female ratio was 2:1. All but one of the patients
(1 Cypriot) were of British origin. Six of the nine patients had holidayed in the endemic re-
gions, and three worked or had a holiday home there. The median time to diagnosis was six
months, range of 3–12 months.
Leishmania amastigotes were seen in biopsies taken from all immunocompetent patients
(six bone marrow aspirates, one subcutaneous nodule, one splenic biopsy and one splenic aspi-
rate). Serology was performed on seven of the patients, all of whom were positive on DAT and
k39 antigen. In three patients, the causative species of protozoa was identified by DNA PCR as
Leishmania donovani.
All patients received liposomal amphotericin B. Eight of the nine immunocompetent pa-
tients were cured. One patient developed mucocutaneous leishmaniasis (ML)13 years later (VL
diagnosis 1996, ML diagnosis 2009) requiring treatment with sodium stibogluconate and was
then cured.
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Immunocompromised Patients
There were 19 patients in the immunocompromised category. Seven patients were HIV posi-
tive and five were on highly active anti-retroviral therapy (HAART) when their VL was diag-
nosed. See Table 1 for a summary of these patients.
Six patients had autoimmune disease, all of whom were taking immunomodulatory drugs at
the time of diagnosis. Two further patients had haematological malignancies: chronic lym-
phoid leukaemia and T cell lymphoma. Of the remaining patients, their immunocompromise
may have been attributable to diabetes, chronic alcohol excess or multiple co-morbidites. See
Table 2 and S1 Fig for further information on these patients.
Fig 1. a: Areas of Acquisition for whole cohort. b: Countries of Acquisition in Mediterranean Basin for whole cohort.
doi:10.1371/journal.pone.0121418.g001
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11 patients in the immunocompromised group were British. The remaining eight were from
Spain, Italy, India, Algeria, Ethiopia and Eritrea. The country of acquisition of VL was predom-
inantly in the Mediterranean basin, whilst three were in Africa and two in India. Ten patients
holidayed in the endemic zone, four patients had a holiday home, two were visiting friends and
relatives, two had migrated to the endemic zone and one patient was there to work. Median age
at diagnosis was 48.5 years, male: female ratio was 2:1. The median time to diagnosis was five
months with a range of 1–120 months.
Table 1. Patients with retroviral disease.
Therapy and markers at time of VL diagnosis ARV Alteration Relapse
1 CD4 count = 269 cells/ mm
3
Saquinavir 1g BD Ritonavir
100mg BD Tenofovir 245mg OD Namivudine 150mg BD
ARV changed by HIV team to Saquinavir,
Ritonavir, Truvada (emtricitabine/ tenofovir).
VL relapsed: treated with ambisome,
miltefosine and pentamidine
prophylaxis.
2 CD4 count = 143 cells/ mm
3
VL <40/ml Darinuvir 800mg OD
Ritonavir 100mg OD
No change in ARV. VL relapsed: treated with ambisone.
3 CD4 count = 140 cells/ mm
3
VL 80/ml Kivexa 1 OD
(abacavir/lamivudine) Kaletra 2 BD (Lopinavir/ritonavir) Patient
also has steroid induced diabetes; HbA1c = 9
No change in ARV. VL relapsed: successfully treated
with ambisone.
4 CD4 count = 120 cells/ mm
3
VL 40/ml Atripla (efavirenz/
emtricitabine/ tenofovir) Ritonavir
ARV’s changed due to rising viral load and
nevirapine resistance. Started Darunavir
and Truvada.
No VL relapse.
5 CD4 count = 410 cells/ mm
3
VL 700 000/ml Kaletra 2 BD No change in ARV. No VL relapse.
6 CD4 count = not available Not on ARVs No VL relapse
7 CD4 count = not available Not on ARVs Relapsed with Post Kala azar dermal
Leishmaniasis Discharged
HIV-1 protease inhibitors (PI) are suggested for inclusion in patients co-infected with HIV and VL. Note all patients on ARV’s were already on Ritonavir
(PI) at time of VL diagnosis.
doi:10.1371/journal.pone.0121418.t001
Table 2. Patients with Immunocompromise not attributable to retroviral disease.
Diagnosis Therapy Relapse
1Graves disease, autoimmune haemolytic anaemia,
diabetes mellitus type 2 (HbA1c unknown).
Prednisolone 10mg OD Azathioprine 50mg OD Metformin
(dose unknown)
No
2Systemic lupus erythematosus Prednisolone 6mg OD Hydroxychloroquine 400mg BD No
3Rheumatoid arthritis Methotrexate (dose unknown). Stopped at time of VL
diagnosis
No
4Rheumatoid arthritis Methotrexate (dose unknown) Steroids (unknown) No
5Psoriatic arthritis Methotrexate (dose unknown) TNF-alpha inhibitor (dose
unknown)—stopped when patient acutely unwell.
Yes
6Psoriasis, CD4 lymphopaenia Developed ocular
leishmaniasis.
Prednisolone 10mg OD Dapsone 50mg OD Yes, patient still
receiving pentamidine.
7Diabetes mellitus type 2 (HbA1c unknown), alcohol induced
pancreatitis (alcohol intake unknown).
Gliclazide and Metformin (doses unknown). No
8Diabetes mellitus type 2, cervical carcinoma, chronic
kidney disease, ischemic heart disease, CD4
lymphopaenia
Polypharmacy—no immunomodulatory treatment. Yes, discharged after
cure
9Chronic lymphoid leukaemia Unknown medications. No
10 Unspecified T cell lymphoma Prednisolone 10mg OD. Yes
11 Alcohol excess—80 units per week. No
12 Alcohol excess—252 units per week, basal cell carcinoma, CD4 lymphopaenia Yes, on prophylaxis.
doi:10.1371/journal.pone.0121418.t002
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Fourteen patients had Leishmania amastigotes identified on microscopy, twelve in bone
marrow aspirates and two in splenic aspirates. Eight types of tissues were examined and Leish-
mania amastigotes found (four bone marrow, three duodenal, two gastric, two skin, one colon,
one spleen, one lymph node and one liver). One patient was diagnosed on serological testing.
L. donovani was identified as the infecting species by DNA PCR in15 patients from tissue sam-
ples. For a summary of the diagnostic investigations please refer to Table 3.
Most patients (n = 18) were treated with liposomal amphotericin B. Nine patients relapsed
and needed further treatment. Of these nine patients, six were then treated with intermittent
prophylactic medication (five pentamidine, one liposomal amphotericin B).
Of the nine patients who relapsed the immunocompromised subgroups were as follows:
four patients with HIV infection, two patients with psoriatic arthropathy, one patient with
multiple co mobidities including diabetes mellitus, one with a T cell lymphoma and one with a
history of excess alcohol.
Three patients were subsequently found to have a CD4 lymphompenia. See Table 2.
Nine of the 19 were cured and discharged (no longer seen at the HTD for their VL >1 year).
Four patients died during the study period. One died from complications of a VL relapse
which resulted in sepsis and ARDS. One patient died from chronic lymphoid leukaemia and
for the remaining two patients the cause of death was not available. Six remain under the care
of the HTD either on VL prophylaxis or for continued surveillance of their VL. For a summary
of results for the whole cohort please refer to Table 4.
Discussion
The strengths of this study are the complete data set which is a representative cohort from the
HTD. Retrospective data collection and being unable to capture all patients in the UK with VL
during this time period are limitations.
Up to two patients with VL are seen at the HTD annually.[3] The majority of patients are
British tourists travelling to the Mediterranean basin. Our new finding is that patients with
medical immunosuppression are now a significant group at risk of developing VL.
Two thirds of the cohort examined had some form of immunocompromise. HIV positive
patients comprise the largest subgroup of immunocompromised patients at 41% of the cohort,
followed by patients with autoimmune diseases (35%). This is the first report of an association
between VL and autoimmune diseases in a cohort. We found a change in the type of patients
who are developing VL compared to previous publications. [4] The proportion of patients with
VL and immunocompromise has increased with the biggest increase in the autoimmune dis-
ease group. The decline in the proportion with HIV may be due to the widespread use of highly
active anti-retroviral therapy. [15] The increase in patients with autoimmune diseases and VL
may result from more patients with chronic autoimmune diseases being able to tolerate immu-
nomodulation and to travel to endemic zones. [16,17]
The two groups had a similar median time to diagnosis however the absolute ranges were
very different. Further to this the median age at diagnosis in the immunocompromised group
was over ten years older than the immunocompetent group (38 years, 48.5 years). These
Table 3. Outcomes of diagnostic tests per immune status (Positive result/total number of test performed).
Microscopy Histology Serology DNA PCR Culture
Immunocompetent 3/3 6/6 7/7 3/5 0/3
Immunocompromised 14/16 9/9 10/11 15/18 3/11
doi:10.1371/journal.pone.0121418.t003
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findings reflect the diagnostic difficulty posed by VL in the immunocompromised patient
whose presentations can be atypical.
Of the nine immunocompromised patients who relapsed, three were found to have a CD4
lymphopenia. All of these patients remained lymphopenic after treatment and suffered re-
lapses. Two patients developed disseminated disease where amastigotes were found in atypical
locations such as the gastrointestinal tract and in ocular tissues. No other patients in the cohort
developed new lymphopenia after treatment for VL.
The patients with CD4 lymphopenia were identified at the time of their VL diagnosis. We
have no record of their lymphocyte counts prior to this and therefore are unable say if the low
lymphocyte count was the risk factor for developing VL or a consequence of the disease itself.
However despite treatment and cure in one of these patients their lymphocyte counts have re-
mained low which could suggest that the lymphopenia predated the VL. The final two patients
remain on VL prophylaxis.
We know that diabetes and chronic alcohol excess can impair the immune system by im-
pacting on T lymphocyte function [18,19]. Despite limited evidence of the link between these
risk factors and the development of VL, because T cells are critical in controlling leishmania
protozoa, an absolute reduction in lymphocyte number as well as impaired function of the re-
maining lymphocytes could contribute to the development and relapse of VL. For example,
within our cohort, two patients with CD4 lymphopenia and either diabetes or alcohol excess
developed VL relapses. However, the small patient numbers mean that diabetes and alcohol ex-
cess remain only potential risk factors for the development of a severe VL course.
We would recommend that a differential lymphocyte count with CD4 cell subsets be includ-
ed in the routine work up for patients who are diagnosed with VL.
Table 4. Summary of Results.
Immunocompetent patients (n = 9) Immunocompromised patients (n = 19)
Demographics Median age at diagnosis = 38 M:F = 2:1. 8 British, 1 Cypriot Median age at diagnosis = 48.5 years M:F = 2:1 11 British, 1
Spanish, 1 Italian, 1 Indian, 1 Algerian, 1 Ethiopian, and 1
Eritrean. (2 patients country of origin not recorded)
Reason in
endemic zone
6 Holiday 2 holiday home 1 work 10 Holiday; 4 holiday home; 2 visiting friends and relatives; 2
migrated to the endemic zone; 1 for work
Median time to
diagnosis
6 months, range of 3–12 months 5 months, range of 1–120 months
Diagnostic
method
Microscopy: 6 bone marrow biopsy, 1 subcutaneous nodule, 1
splenic biopsy, 1 splenic aspirate. Serology:7 positive DAT and
k39 antigen. DNA PCR: L. donovani identified in 3 patients
Microscopy: 12 in bone marrow aspirates, 2 in splenic aspirates,
8 types of tissues were examined and Leishmania amastigotes
found: bone marrow 4, duodenal 3, gastric 2, skin 2, colon 1,
spleen 1, lymph node 1 liver. Serology:1 patient was diagnosed
on serological testing. DNA PCR: L. donovani identified in 15
patients
Treatment All received liposomal amphotericin B 18 treated with liposomal amphotericin B. 9 patients relapsed
requiring further treatment 6 patients needed intermittent
prophylactic Medication. Pentamidine Prophylaxis Patient 1:3
weekly for 2 years, Patient 2:2–3 weekly for >6 years, Patient 3:
Monthly recorded for 1 year, Patient 4:Monthly, unknown
duration, Patient 5:3 weekly recorded for 1 year, Liposomal
amphotericin B Prophylaxis, Patient 6: monthly
Outcome 8 of the 9 were cured and discharged. 1 later developed
mucocutaneous leishmaniasis, was treated with sodium
stibogluconate and was then cured.No deaths recorded.
9 of the 19 were cured and discharged. 4 died during the study
period. 1 patient died from complications of a VL relapse, which
resulted in sepsis and ARDS. 1 patient died from chronic
lymphoid leukaemia and for the remaining 2 patients the cause of
death was not available. 6 remain under the care of the HTD on
VL prophylaxis or for continued surveillance of their VL.
doi:10.1371/journal.pone.0121418.t004
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We would also suggest raising awareness of VL within the rheumatological community and
that VL be considered as part of the differential diagnosis in rheumatology patients with pyrex-
ia of unknown origin who return from travel to endemic areas.
Supporting Information
S1 Fig. Timeline of VL Diagnosis in Immunocompromised Patients.
(TIFF)
Acknowledgments
We thank Margaret Armstrong, clinical research coordinator at The HTD, for continued ad-
ministrative support throughout the project. Thanks to Julie Watson (Department of Clinical
Parasitology, Hospital for Tropical Diseases) for technical contributions to the paper. This
study was supported by the Special Trustees of the Hospital for Tropical Diseases. Margaret
Armstrong is supported by The Special Trustees of the Hospital for Tropical Diseases.
Author Contributions
Conceived and designed the experiments: DL. Performed the experiments: DL. Analyzed the
data: KF RI DL. Contributed reagents/materials/analysis tools: KF RI DL. Wrote the paper: KF
RI DL. Edited manuscript: KF RI DL. Designed data collection proforma and data collection:
KF RI.
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