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Toll-like Receptor 4 Polymorphisms and Aspergillosis

To the Editor: Bochud et al. (Oct. 23 issue)1
present data suggesting an association between
toll-like receptor 4 (TLR4) haplotypes in unrelated donors and an increased risk of invasive aspergillosis among recipients of allogeneic hematopoietic-cell transplants. The authors theorize that
this association is due to the capacity of TLR4 to
recognize ligands present on Aspergillus fumigatus.2 However, there is another possible explanation for the findings that should be considered.
During much of the period of the study, transplant recipients with febrile neutropenia that was
unresponsive to antibiotics received amphotericin
B. This is significant because conventional and
lipid formulations of amphotericin B activate
phagocytic cells by stimulating toll-like receptor 2
(TLR2) and TLR4 to release proinflammatory cytokines and chemokines.3,4 These immunomodulatory properties of amphotericin B may contribute to its antifungal activity.5 Optimal
efficacy of liposomal amphotericin B in mice
with bone marrow transplants and aspergillosis
has been linked to TLR4-mediated activation of
neutrophils by amphotericin B.4 Thus, the association between donor TLR4 haplotypes and the
this weeks letters
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Toll-like Receptor 4 Polymorphisms

and Aspergillosis

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Clostridium difficile

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Venous Thromboembolic Disease and Pregnancy

640

Prevalence of Mitochondrial 1555AG Mutation

in European Children

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Prevalence of Mitochondrial 1555AG Mutation

in Adults of European Descent

risk of aspergillosis may be due at least in part to

decreased immunostimulation of donor cells by
amphotericin B.
Stuart M. Levitz, M.D.
University of Massachusetts Medical School
Worcester, MA 01605
stuart.levitz@umassmed.edu

Shmuel Shoham, M.D.

Washington Hospital Center
Washington, DC 20010

John D. Cleary, Pharm.D.

University of Mississippi Medical Center
Jackson, MS 39216
1. Bochud P-Y, Chien JW, Marr KA, et al. Toll-like receptor 4

polymorphisms and aspergillosis in stem-cell transplantation.

N Engl J Med 2008;359:1766-77.
2. Mambula SS, Sau K, Henneke P, Golenbock DT, Levitz SM.
Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus. J Biol Chem 2002;277:39320-6.
3. Sau K, Mambula SS, Latz E, Henneke P, Golenbock DT, Levitz SM. The antifungal drug amphotericin B promotes inflammatory cytokine release by a Toll-like receptor- and CD14-dependent mechanism. J Biol Chem 2003;278:37561-8.
4. Bellocchio S, Gaziano R, Bozza S, et al. Liposomal amphotericin B activates antifungal resistance with reduced toxicity by
diverting Toll-like receptor signalling from TLR-2 to TLR-4.
J Antimicrob Chemother 2005;55:214-22.
5. Rogers PD, Pearson MM, Cleary JD, Sullivan DC, Chapman
SW. Differential expression of genes encoding immunomodulatory proteins in response to amphotericin B in human mononuclear cells identified by cDNA microarray analysis. J Antimicrob
Chemother 2002;50:811-7.

To the Editor: Bochud and colleagues demonstrate an important association of TLR4 polymorphisms with aspergillosis after allogeneic stemcell transplantation. As the authors point out, TLR4
polymorphisms might be associated with other
risk factors for aspergillosis. Cytomegalovirus
(CMV) disease is strongly and independently associated with aspergillosis. In a recent large study
of stem-cell transplant recipients, patients with
CMV disease had the highest overall hazard ratio

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correspondence

for aspergillosis (6.9), and the hazard ratio was

higher with an early onset of CMV disease than
with a late onset.1 Although TLR4 does not seem
to be directly involved in the recognition of CMV,
TLR4 ligands enhance the ability of dendritic cells
to present CMV antigen, resulting in an increased
number of antigen-specific CD4+ and CD8+ cells.2
Two studies have shown an association of TLR4
polymorphisms with CMV disease after renal transplantation.3,4 Therefore, CMV could be an intermediate variable in the association of TLR4 polymorphisms with aspergillosis, and it may be
questionable to consider it as a confounding factor in a regression model. The important findings reported by Bochud and colleagues would
be strengthened by an analysis of the association
between TLR4 polymorphisms and CMV disease.
Carlos Cervera, M.D.
Asuncion Moreno, Ph.D.
Francisco Lozano, Ph.D.
Hospital Clinic of Barcelona
08023 Barcelona, Spain
ccervera@clinic.ub.es
1. Garcia-Vidal C, Upton A, Kirby KA, Marr KA. Epidemiology

of invasive mold infections in allogeneic stem cell transplant

recipients: biological risk factors for infection according to time
after transplantation. Clin Infect Dis 2008;47:1041-50.
2. Lor K, Betts MR, Brenchley JM, et al. Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirus- and
HIV-1-specific T cell responses. J Immunol 2003;171:4320-8.
3. Cervera C, Lozano F, Saval N, et al. The influence of innate
immunity gene receptors polymorphisms in renal transplant infections. Transplantation 2007;83:1493-500.
4. Ducloux D, Deschamps M, Yannaraki M, et al. Relevance of
Toll-like receptor-4 polymorphisms in renal transplantation.
Kidney Int 2005;67:2454-61.

To the Editor: The single-nucleotide polymorphism in the third exon of human TLR4 (Asp299Gly)
and the cosegregating missense mutation at amino
acid 399 (Thr399Ire), which correspond to the
extracellular domain of TLR4, have different distributions in different populations. Whereas 5 to
20% of persons in white populations bear these
cosegregating TLR4 polymorphisms, they are virtually missing in Asian populations.1 Nonetheless,
severe invasive aspergillosis develops in many patients undergoing allogeneic hematopoietic stemcell transplantation in Japan,2 which raises a question regarding the extent to which the risk of
invasive aspergillosis among patients undergoing
allogeneic hematopoietic stem-cell transplantation
is attributable to the donor haplotype of these mutations. To prove their hypothesis, Bochud et al.
must present, in addition to their epidemiologic

results, direct evidence that the cells of the immune system bearing these mutations are actually
hyporesponsive to aspergillus antigens.3
Yusuke Asakura, M.D., Ph.D.
Toru Komatsu, M.D., Ph.D.
Aichi Medical University
Aichi 480-1195, Japan
yasakura@aichi-med-u.ac.jp
1. Ferwerda B, McCall MB, Alonso S, et al. TLR4 polymor-

phism, infectious diseases, and evolutionary pressure during

migration of modern humans. Proc Natl Acad Sci U S A 2007;
104:16645-50.
2. Yamasaki S, Heike Y, Mori S, et al. Infectious complications
in chronic graft-versus-host disease: a retrospective study of 145
recipients of allogeneic hematopoietic stem cell transplantation
with reduced- and conventional-intensity conditioning regimens.
Transpl Infect Dis 2008;10:252-9.
3. Arbour NC, Lorenz E, Schutte BC, et al. TLR4 mutations are
associated with endotoxin hyporesponsiveness in humans. Nat
Genet 2000;25:187-91.

The authors reply: Levitz et al. hypothesize that

the association between donor TLR4 Asp299Gly
and the risk of aspergillosis may be due to decreased immunostimulation of donor cells by amphotericin B. This hypothesis is not supported by
a recent study by Carvalho et al., which linked the
Asp299Gly polymorphism to chronic cavitary aspergillosis.1 Unlike patients undergoing hematopoietic-cell transplantation, patients in whom chronic cavitary aspergillosis develops usually do not
receive amphotericin B before the diagnosis of
fungal infection is made. Thus, it is most likely
that polymorphisms in TLR4 can influence susceptibility to aspergillosis in a manner that is
independent of administered amphotericin B.
Cervera et al. suggest that the TLR4 Asp299Gly
may be associated with CMV disease, which
would in turn lead to increased susceptibility to
invasive aspergillosis. This hypothesis is not supported by the multivariate analysis performed in
our validation study, which included CMV disease
as a covariate and identified the TLR4 polymorphisms as an independent risk factor for inva
sive aspergillosis. Also, the association between
Asp299Gly and chronic cavitary aspergillosis suggests that there may be a more direct association
with diseases caused by aspergillus species.1 Although an association between TLR4 polymorphisms and CMV disease cannot be ruled out, this
study suggests that TLR4 polymorphisms influence susceptibility to aspergillosis in a manner
that is independent of CMV disease.
The role of ethnicity in susceptibility to infec-

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The

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tions is an interesting topic, as raised by Asakura Pierre-Yves Bochud, M.D.

and Komatsu. In contrast to primary immunode- Centre Hospitalier Universitaire Vaudois
ficiencies, which typically involve a single, rare 1011 Lausanne, Switzerland
polymorphism, leading to important functional Kieren A. Marr, M.D., Ph.D.
consequences, susceptibility to infections in the Johns Hopkins University
general population is usually considered to result Baltimore, MD 21205
from variants in several genes, each leading to Michael Boeckh, M.D.
smaller functional alterations. Results of in vitro Fred Hutchinson Cancer Research Center
and ex vivo studies have shown that innate im- Seattle, WA 98109
mboeckh@fhcrc.org
mune recognition of fungal pathogens is mediated by several receptors a finding that is not 1. Carvalho A, Pasqualotto AC, Pitzurra L, Romani L, Denning
surprising, given the complicated cell-wall archi- DW, Rodrigues F. Polymorphisms in toll-like receptor genes and
tecture of fungi.2 In a Korean population, a haplo- susceptibility to pulmonary aspergillosis. J Infect Dis 2008;197:
618-21.
type in the interleukin-10 gene (IL-10) was reported 2. Netea MG, Brown GD, Kullberg BJ, Gow NA. An integrated
to influence susceptibility to invasive aspergillo- model of the recognition of Candida albicans by the innate imsis3; also, a recent study identified a polymorphism mune system. Nat Rev Microbiol 2008;6:67-78.
3. Seo KW, Kim DH, Sohn SK, et al. Protective role of interleuin the host plasminogen gene that was associ- kin-10 promoter gene polymorphism in the pathogenesis of in4
ated with a risk of invasive aspergillosis. Thus, vasive pulmonary aspergillosis after allogeneic stem cell transwe believe that inherited risks are likely to be plantation. Bone Marrow Transplant 2005;36:1089-95.
4. Zaas AK, Liao G, Chien JW, et al. Plasminogen alleles influmultifactorial and to differ among specific ethnic ence susceptibility to invasive aspergillosis. PLoS Genet 2008;
groups, with polymorphisms in genes other than 4(6):e1000101.
TLR4 also influencing susceptibility to invasive
infection and airway disease.

Clostridium difficile
To the Editor: I would add two comments to
Kelly and LaMonts review of the management of
Clostridium difficile infection (Oct. 30 issue).1 First,
in the diagnosis of C. difficile infection, it is important to note that the positive predictive value
of commercially available assays for the detection
of C. difficile toxin is unacceptably low, and a twostage testing strategy for C. difficile with an initial
sensitive rapid screening test and confirmation
of positive samples by a reference method has recently been recommended.2 Second, the benefit
of anion-exchange resins such as cholestyramine
in the treatment of recurrent C. difficileassociated
diarrhea was first recognized in the early 1980s
and should not be forgotten as a management
option.3

To the Editor: In commenting on the rising incidence of C. difficile infection, Kelly and LaMont
make no mention of the association between the
use of proton-pump inhibitors and the risk of
C. difficileassociated disease. Proton-pump inhibitors are among the most widely prescribed medications in the world. The inhibition of gastric acid
removes the normal defense mechanism against
ingested bacteria and spores.1 Gastric acid suppression by proton-pump inhibitors is associated
with an increased risk of enteric infection, especially with campylobacter, salmonella,2 and C. difficile.1,3-5 The use of proton-pump inhibitors could
be an important factor in the increasing incidence
of C. difficile infection.
Sammy A. Baierlein, M.D.

Richard C.G. Pollok, Ph.D.

Ligusterstrasse 20
95488 Eckersdorf, Germany
sammybaierlein@web.de

St. Georges University of London

London SW17 0QT, United Kingdom
1. Kelly CP, LaMont JT. Clostridium difficile more difficult

than ever. N Engl J Med 2008;359:1932-40.

2. Planche T, Aghaizu A, Holiman R, et al. Diagnosis of Clos-

tridium difficile infection by toxin detection kits: a systematic

review. Lancet Infect Dis 2008;8:777-84.
3. Viscidi RP, Bartlett JG. Antibiotic-associated pseudomembranous colitis in children. Pediatrics 1981;67:381-6.

636

Anja Wistop, M.D.

Klinikum Kulmbach
95326 Kulmbach, Germany
1. Yearsley KA, Gilby LJ, Ramadas AV, Kubiak EM, Fone DL,

Allison MC. Proton pump inhibitor therapy is a risk factor for

Clostridium difficile-associated diarrhoea. Aliment Pharmacol
Ther 2006;24:613-9.

n engl j med 360;6 nejm.org february 5, 2009

The New England Journal of Medicine

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Copyright 2009 Massachusetts Medical Society. All rights reserved.