Parasitol Res

DOI 10.1007/s00436-016-5313-x

REVIEW

Prevalence, severity, and pathogeneses of anemia

in visceral leishmaniasis
Yasuyuki Goto 1 & Jingjie Cheng 1,2 & Satoko Omachi 1 & Ayako Morimoto 1

Received: 13 September 2016 / Accepted: 26 October 2016

# Springer-Verlag Berlin Heidelberg 2016

Abstract Anemia is a typical symptom during visceral leish- Introduction

maniasis (VL). We performed a systematic analysis of the
literature on anemia in VL to understand the prevalence, se- Anemia is clinically defined by a decrease in hemoglobin (Hb)
verity, and possible mechanisms. Anemia is very common in levels below the reference level based on age and sex of the
VL patients with an overall prevalence higher than 90 %. The individual, when other factors, such as pregnancy and altitude,
degree of anemia in VL is moderate to severe (hemoglobin are taken into account (World_Health_Organization 2001).
level ∼7.5 g/dl), and the status can be recovered by treatment According to the World Health Organization (WHO), in hu-
with antileishmanial drugs within a certain period of time. man adults, a Hb level below 13 g/dl in men and 12 g/dl in
Possible pathogeneses of anemia in VL based on clinical ob- women is considered anemic (World_Health_Organization
servations included anti-RBC antibodies, dysfunction in 2011). Broadly speaking, the causes of anemia can be classi-
erythropoiesis, and hemophagocytosis in the bone marrow fied into three categories: red blood cell (RBC) loss, reduced
or spleen, while hemolysis is a more likely cause than RBC production, and destruction of RBC in the body.
dyserythropoiesis. In hamsters with experimental VL, Worldwide, iron deficiency is the most common cause of
hemophagocytosis induced by immune complex and changes anemia (McLean et al. 2009). Iron is required for Hb synthe-
on erythrocyte membrane is speculated as the pathogenesis for sis; if iron stores are low due to reasons ranging from malab-
anemia. In contrast, our recent study on murine VL indicated sorption to menorrhagia, RBC production is unable to keep up
that hemophagocytosis contributes to anemia in contrast to with the body’s needs. Other causes of reduced RBC produc-
lower contribution of anti-RBC antibodies or dysfunction in tion include hemoglobinopathies such as thalassemia, as well
erythropoiesis. Together, hemophagocytosis is most likely as- as neoplasia. Hemolytic anemia, on the other hand, can be
sociated with anemia in VL, and elucidation of the immuno- intravascular or extravascular. While several genetic condi-
logical mechanisms may lead to development of novel inter- tions predispose individuals to acute intravascular hemolytic
ventions to manage the symptom. episodes, these episodes are often triggered by external insults
such as an infection or certain foods. Examples include dis-
seminated intravascular coagulation as a complication of sick-
Keywords Visceral leishmaniasis . Anemia . Hemolysis . le cell anemia and hemolytic episodes in individuals with
Hemophagocytosis enzyme deficiencies caused by oxidative insults (van Wijk
and van Solinge 2005). Causes of extravascular hemolytic
anemia include autoimmune hemolytic anemia (AIHA) and
* Yasuyuki Goto hereditary spherocytosis (Gallagher 2010). In AIHA, IgG an-
aygoto@mail.ecc.u-tokyo.ac.jp tibodies react with protein antigens on RBCs while hereditary
spherocytosis is a result of intrinsic defects in RBC membrane
1
Laboratory of Molecular Immunology, Department of Animal proteins resulting in cytoskeleton instability.
Resource Sciences, Graduate School of Agricultural and Life Human visceral leishmaniasis (VL) is a vector-borne dis-
Sciences, The University of Tokyo, Tokyo, Japan ease caused by the leishmanial species Leishmania donovani
2
Faculty of Medicine, Imperial College London, London, England and Leishmania infantum. VL is the most severe form of
 Parasitol Res

leishmaniasis with an estimated 200,000 to 400,000 new cases 2010; Tanir et al. 2006; Tanoli et al. 2005; van den Bogaart
and 20,000 to 40,000 deaths annually (WHO/ et al. 2012; van den Bogaart et al. 2013; Zijlstra et al. 1991).
Regional_Office_for_South-East_Asia 2013). It is character- The mean prevalence of anemia in three studies using Hb
ized by a clinical picture that includes hepatosplenomegaly, levels of 11 g/dl as the cutoff was 98.8 % (range, 96.5–100
chronic fever, weight loss, and anemia, after an incubation %) (Haidar et al. 2001; Tanir et al. 2006; van den Bogaart et al.
period lasting between 2 and 6 months (Chappuis et al. 2012). Two other studies using similar cutoffs also presented
2007). As the hematological disorder results in lower ability high prevalence of anemia (100 % at <10.5 g/dl and 92 % at
to supply oxygen to the tissues, anemia may play a central role ≤10 g/dl) (Elnour et al. 2001; Zijlstra et al. 1991), demonstrat-
in causing other symptoms found in VL patients including ing that prevalence of anemia is very high in VL patients. Six
fatigue, malaise, weakness, and pallor. studies described the prevalence of anemia for the Hb levels <
While anemia is a common sign in parasitic infections, or ≤9 g/dl with the mean of 83.2 % (range, 75.0–87.9 %)
the proposed mechanisms are variable. In this review, we (Blazquez-Gamero et al. 2015; Bode et al. 2014; Braga et al.
performed a systematic review of the literature on anemia 2013; Cachia and Fenech 1964; Cascio et al. 2002; Elnour
in VL to precisely understand the prevalence and severity et al. 2001). Four studies using a cutoff value of <7 g/dl had
of VL-associated anemia as well as the speculated the mean prevalence of anemia as 52.8 % (range, 27.7–83.3
mechanisms. %) (Brustoloni et al. 2010; Costa et al. 2010; Gagnaire et al.
2000; Lahlou et al. 2011). These results suggest that anemia
during VL is often severe.
Prevalence and severity of anemia in VL Thirty-four studies described the mean, median, or
range of Hb levels in VL patients (Fig. 1) (Ansar et al.
The PubMed search of the literature was performed using 2009; Arik Yilmaz et al. 2009; Blazquez-Gamero et al.
Bvisceral leishmaniasis^ plus Banemia^ or Bhematology^ as 2015; Bode et al. 2014; Cascio et al. 2002; Castagnola
the search terms. As of March 10, 2016, The PubMed search et al. 1996; Collin et al. 2004; Gagnaire et al. 2000;
found 309 articles with Bvisceral leishmaniasis^ plus Banemia,^ Grech et al. 2000; Lahlou et al. 2011; Lal et al. 2016;
90 articles with Bvisceral leishmaniasis^ plus Bhematology,^ Lita et al. 2002; Madalosso et al. 2012; Minodier et al.
and 386 different articles in total. Among them, some articles 1998; Mueller et al. 2014; Mukherjee et al. 2012; Nail
were excluded from the analysis if (a) the article was not about and Imam 2013; Petrela et al. 2010; Rahim and Ashkan
human cases, (b) the case number in the article was less than 10, 2007; Saeed et al. 1998; Samanta et al. 2011; Samanta
(c) the article was not written in English, or (d) the article did not et al. 2012; Singh et al. 1999; Souza et al. 2012; Sundar
describe anemic status in VL patients. As a result, 55 articles et al. 2010; Sundar et al. 2002; Tanir et al. 2006; Tavora
matched the criteria for the analysis. et al. 2015; Thakur et al. 1999; Tofighi Naeem et al. 2014;
Among them, 38 studies described the prevalence of ane- van den Bogaart et al. 2012; van den Bogaart et al. 2013;
mia in VL patients (Arik Yilmaz et al. 2009; Basset et al. Vilela et al. 2002; Zijlstra et al. 1991). The majority of the
2005; Blazquez-Gamero et al. 2015; Bode et al. 2014; Braga studies had the mean/median value between 6 and 8 g/dl, dem-
et al. 2013; Brustoloni et al. 2010; Cachia and Fenech 1964; onstrating anemia in VL is moderate to severe according to the
Cascio et al. 2002; Castagnola et al. 1996; Chandra et al. 2013; WHO criteria (World_Health_Organization 2011). Two studies
Costa et al. 2010; Dursun et al. 2009; Elnour et al. 2001; describing anemia in HIV-infected VL patients and their Hb
Evans et al. 1985; Gagnaire et al. 2000; Haidar et al. 2001; levels were in a similar range to non-HIV VL patients (Souza
Hamzavi et al. 2012; Hassan et al. 1995; Jeannel et al. 1991; et al. 2012; Tavora et al. 2015), suggesting HIV infection has no
Lahlou et al. 2011; Lita et al. 2002; Mengesha et al. 2014; big impact on anemia during VL. Seven studies described ki-
Minodier et al. 1998; Nail and Imam 2013; Petrela et al. netics of Hb levels in VL patients over the course of chemo-
2010; Rahim et al. 1998; Rosenthal et al. 2000; Sampaio therapy (Castagnola et al. 1996; Mukherjee et al. 2012; Saeed
et al. 2010; Sarkari et al. 2016; Souza et al. 2012; Tanir et al. et al. 1998; Sundar et al. 2010, 2002; Thakur et al. 1999; Vilela
2006; Tanoli et al. 2005; Tavora et al. 2015; Tofighi Naeem et al. 2002), indicating that the anemic status can be recovered
et al. 2014; van den Bogaart et al. 2012; van den Bogaart et al. by treatment with antileishmanial drugs with certain period of
2013; Vilela et al. 2002; Zijlstra et al. 1991), whereas only 21 time (Fig. 2).
of them clearly showed the diagnostic criteria of anemia based
on Hb levels or hematocrit (Arik Yilmaz et al. 2009;
Blazquez-Gamero et al. 2015; Bode et al. 2014; Braga et al. Possible causes of anemia in VL patients
2013; Brustoloni et al. 2010; Cachia and Fenech 1964; Cascio
et al. 2002; Costa et al. 2010; Elnour et al. 2001; Evans et al. While VL patients may present with a range of hematological
1985; Gagnaire et al. 2000; Haidar et al. 2001; Lahlou et al. abnormalities including leucopenia, thrombocytopenia, and
2011; Nail and Imam 2013; Petrela et al. 2010; Sampaio et al. pancytopenia, anemia is the most common (Varma and
Parasitol Res

Fig. 1 Anemia in VL is moderate

to severe. Mean (circle), median
(square), or range (bar) of Hb
levels in VL patients (black),
treated VL patients (red), or
controls (blue) are shown. Green
symbols represent VL patients
with confirmed HIV infection.
Error bars represent SD

Naseem 2010). Numerous mechanisms have been proposed However, the frequency of bleeding among VL patients is less
for the mechanism of anemia in VL, in all three categories of than 10% in most cases (Coura-Vital et al. 2014; Dhingra et al.
blood loss, reduced RBC production, and increased hemoly- 2010; Lita et al. 2002; Sampaio et al. 2010) and is much lower
sis. Here, we review the causes of anemia in VL based on than that of anemia. Therefore, it is unlikely that bleeding is
human cases as well as findings in experimental VL. the major cause of anemia during VL, whereas it might be
associated with severe anemia leading to death.

Blood loss Dysfunction in erythropoiesis

There is a case report that a 7-month-old girl with fever, bleed- Bone marrow is the major tissue for production of erythrocytes
ing from the mouth, and anemia was later confirmed for in humans and is also one of the major sites for Leishmania
Leishmania infection (Chaliasos et al. 1996). There are also parasitism. Therefore, it is not surprising to have
some reports on retinal bleeding, digestive bleeding, or epi- dyserythropoiesis in the bone marrow during VL (Sheikha
staxis in VL patients (De Cock et al. 1982; Desoubeaux et al. 2004; Wickramasinghe et al. 1987; Yarali et al. 2002), since
2015; Laguna et al. 1994; Montero et al. 2003). Because Leishmania infection often causes changes in cell types in the
thrombocytopenia is also a typical hematological disorder in tissue. Fibrosis in bone marrow has been reported as associated
VL, it is not strange that VL patients have tendency for bleed- with VL (Dhingra et al. 2010; Rocha Filho et al. 2000; Saleem
ing. In fact, bleeding is a risk factor for death from VL (Costa et al. 1991). However, the significance of bone marrow fibrosis
et al. 2010; Coura-Vital et al. 2014; Sampaio et al. 2010). on anemia in VL is uncertain. First, one of the reports demon-
strated no difference in Hb levels of VL patients with or without
bone marrow fibrosis (Rocha Filho et al. 2000). Second,
hematological/biochemical parameters indicate rather up-
regulated erythropoiesis in VL patients. Serum erythropoietin
levels were high in VL patients compared with control subjects,
showed a negative correlation with Hb levels, and decreased
over the course of chemotherapy (Saeed et al. 1998).
Correspondingly, increased reticulocyte counts or erythroid hy-
perplasia are often found in VL patients (Rahim and Ashkan
2007; Samanta et al. 2011; Wickramasinghe et al. 1987).
Other possible cause in dyserythropoiesis during VL is iron
deficiency (Elnour et al. 2001; Lal et al. 2013; Paul et al. 2014).
Some studies found erythrocytes in VL patients as microcytic
Fig. 2 Recovery from anemia in VL patients by chemotherapy. Kinetics and hypochromic (Cachia and Fenech 1964; Kager and Rees
of Hb levels in VL patients over the course of chemotherapy is shown. 1986). In patients with chronic VL, anemia may be due to
The number and the term in parentheses in a data set title represent the
PubMed ID of the report and the drug used for treatment, respectively.
mechanisms similar to that found in anemia of chronic disease,
LAB liposomal amphotericin B, ABD amphotericin B deoxycholate, Mil such as the abnormal iron retention by macrophages (Pippard
miltefosine, Sb pentavalent antimonials et al. 1986). However, serum iron levels are not consistently
 Parasitol Res

low in VL patients (Mehabresh and el-Mauhoub 1992; Saeed Externalization of phosphatidylserine can also be an indicator
et al. 1998), and normocytic and normochromic anemia is also of damages to erythrocytes and serve as a signal for enhanced
common (Pearson and Sousa 1996), making the significance of phagocytosis during VL (Samanta et al. 2012). Inflammatory
iron deficiency on VL-associated anemia elusive. mediators such as TNF-α and IL-1 are known to decrease
erythrocyte survival (Scharte and Fink 2003). One study re-
Up-regulated destruction of erythrocytes ported that severe anemia in VL patients was related to inflam-
matory signs such as vomiting, edema, and lung inflammation
Hemophagocytosis is a phenomenon that macrophages or histio- (Costa et al. 2010). Together, inflammatory status of VL pa-
cytes engulf erythrocytes and/or leukocytes in the bone marrow, tients may affect erythrocyte life span.
liver, or spleen (Henter et al. 2007). This uncommon AIHA is another possible mechanism for increased hemo-
hemophagocytosis has been reported in autoimmune diseases lysis during VL. The emergence of anti-erythrocyte antibodies
(Kumakura et al. 2004; Tabata et al. 2009) and infectious dis- has been reported in human VL patients (Pontes De Carvalho
eases (Balasubramanian et al. 2008; Granert et al. 1993; La Gruta et al. 1986; Vilela et al. 2002). The ratio of VL patients pos-
et al. 2007; Mallory 1898; Slovut et al. 1996; Vykuntaraju et al. itive for Coombs test/direct agglutination test on erythrocytes
2015). Patients showing hemophagocytosis are often accompa- ranges from 33 to 83% (Elnour et al. 2001; Kager et al. 1984;
nied with other manifestations such as fever, pancytopenia, and Vilela et al. 2002). IgG molecules can be detected on erythro-
splenomegaly, which are diagnostic criteria for hemophagocytic cytes from the patients (Kharazmi et al. 1982; Pontes De
syndrome (HPS) or hemophagocytic lymphohistiocytosis (HLH) Carvalho et al. 1986; Vilela et al. 2002; Woodruff et al.
(Henter et al. 2007). There are reports on hemophagocytosis in 1972), whereas the amount of autoantibodies may not be very
human VL patients (Bhatia et al. 2011; Chandra et al. 2013; high compared with typical AIHA patients (Vilela et al. 2002;
Gagnaire et al. 2000; Kilani et al. 2006; Ozyurek et al. 2005). Woodruff et al. 1972). Other than immunoglobulins, comple-
VL patients representing hemophagocytosis are often diagnosed ments and C-reactive protein are also speculated as host fac-
as HPS since the typical symptoms of VL are also common in tors associated with enhanced hemophagocytosis through in-
HPS (Gagnaire et al. 2000; Granert et al. 1993; Kilani et al. 2006; teraction with erythrocyte membrane (Ansar et al. 2009;
Ozyurek et al. 2005). Hemophagocytes can be cleared after treat- Kager et al. 1984; Kharazmi et al. 1982; Woodruff et al. 1972).
ment with an antileishmanial drug (Gagnaire et al. 2000), indi-
cating a connection between Leishmania infection and
hemophagocytosis. Most of the studies reported Recent advances in animal models of anemia
hemophagocytosis as a result of microscopic examinations of by Leishmania infection
bone marrow aspirates for parasitological diagnosis of VL, and
the frequency of VL patients having hemophagocytosis in the Through the review on anemia in clinical cases of VL, no
tissue ranges from 7 to 75 % (Bhatia et al. 2011; Chandra et al. single factor is found critically responsible for the pathology,
2013; Daneshbod et al. 2010). Although studies are to be per- whereas up-regulated hemolysis is more likely to contribute to
formed, hemophagocytosis in the spleen would be even higher anemia than other mechanisms. Systematic analyses on mech-
because hypersplenism is speculated by splenomegaly typically anisms of anemia in human VL patients are becoming more
found in VL patients (Dos-Santos et al. 2014; Kager and Rees difficult because biopsy of the bone marrow or spleen for the
1986) and because there is a report on erythrocyte destruction in parasitological diagnosis has gradually been replaced with
the spleen (Woodruff et al. 1972). In fact, we found a higher serological diagnosis (e.g., an rK39 dipstick test) due to highly
degree of hemophagocytosis in the spleen of L. donovani-infect- invasive procedure of some risk to the patients and necessity
ed mice compared with that in the bone marrow (Morimoto et al. of professional skills in microscopy. To further understand the
2016). mechanisms, development of animal models for VL-
How can hemophagocytosis be up-regulated during VL? associated anemia is critical.
Increased osmotic fragility of erythrocytes from VL patients Hamsters are regarded as a better model for VL pathology
indicates damages to erythrocytes in VL patients (Ansar et al. than mice because infected hamsters show anemia,
2009; Biswas et al. 1995; Samanta et al. 2012). Altered sur- hepatosplenomegaly, weight loss, and death (Lafuse et al.
face conditions, i.e., binding of C-reactive protein, including 2013; Moreira et al. 2016; Moreira et al. 2012). L. donovani-
lipid peroxidation, glycosylation of spectrin, and 9-O-acetyla- induced anemia has been reported in hamster models, and pro-
tion of sialic acid, can be found on erythrocytes in VL patients posed mechanisms of anemia in hamster studies are
(Ansar et al. 2009; Biswas et al. 1997; Chava et al. 2004; hemophagocytosis induced by immune complex on erythro-
Samanta et al. 2011; Samanta et al. 2012). Although it is not cytes and changes on erythrocyte membrane (Agu et al. 1982;
clear whether these changes are caused or results of damage Biswas et al. 1992; Lafuse et al. 2013). Anemia in L. donovani
on erythrocytes, such abnormality may result in enhanced hamsters is associated with increased serum erythropoietin and
removal of erythrocytes of VL patients at the spleen. decreased serum iron levels (Lafuse et al. 2013). Because
Parasitol Res

Leishmania parasites do not have a heme synthetic pathway unchanged from the uninfected mice. Besides, there was no
(Chang and Chang 1985) and can uptake heme from transferrin detectable level of IgG on the surface of erythrocytes from the
and lactoferrin of the host (Wilson et al. 1994), it is possible that infected mice when tested by a direct agglutination test.
iron deficiency during VL is due to iron usage by the parasites. Furthermore, we found that mice with defect in B cell function
On the other hand, a report demonstrated that production of show a similar degree of anemia to immunocompetent mice by
erythrocytes seems to be intact in the infected hamsters L. donovani infection (Omachi S et al., unpublished data), indi-
(Biswas et al. 1992). Results in hamster models are also incon- cating a limited contribution of antibodies to anemia in this mod-
clusive for the mechanisms of anemia during VL, requiring el. Hemophagocytosis by pathogen-infected macrophages, as
further studies to compare the significance of individual factors observed in our model, has also been reported in Salmonella
side-by-side whereas a paucity of immunological reagents (e.g., infection (Pilonieta et al. 2014) and is a good contrast to that in
antibodies) and animal handling/welfare (e.g., housing, genera- EB virus or Trypanosoma brucei infection where
tion of transgenic animals) would hamper such detailed analy- hemophagocytes are not necessarily infected macrophages
ses. Although mice are more appropriate experimental animals (Brown et al. 2010; Cnops et al. 2015). Therefore, further studies
for such analyses, development of mouse models for VL pa- on hemophagocytosis, with the focus on hemophagocytosing
thologies, especially on anemia, has been lagging behind. macrophages rather than erythrocytes, will facilitate understand-
Recently, we have succeeded for the first time to induce ing of anemia induced by L. donovani infection.
anemia in mice by infection with L. donovani (Morimoto et al. In summary, anemia in VL is moderate to severe, which
2016). In that study, mice at 6 months of infection displayed a means the necessity of careful management. VL patients
∼20% decline in Hb levels compared with uninfected age- with severe anemia often require blood transfusion before
matched controls. Interestingly, the infected mice showed in- starting chemotherapy due to weakness. As steroids and
creased polychromatic erythrocytes/reticulocytes in the circu- etoposide are used for management of symptoms in HLH
lating blood accompanied with increased serum levels of patients including anemia, such interventions to manage
erythropoietin, suggesting increased hemolysis rather than de- anemia may be used for VL patients to improve their QOL
fect in erythropoiesis as a major cause of anemia in the mice. and to increase their tolerance to chemotherapy. Therefore,
In those L. donovani-infected mice, hemophagocytosis was it is important to elucidate the most contributing factor to
prominent in the spleen. An intriguing feature of anemia in VL, despite that it can be multifactorial. Both
hemophagocytosis in murine VL is that the hemophagocytes clinical and experimental data suggest a large contribution
in the spleen were all parasitized and often multinucleated, of hemolysis to causing anemia in VL, and our recent work
suggesting that L. donovani causes phenotypic changes to suggests that hyper-activation of parasitized macrophages is
the infected macrophages towards hyper-activation. In the a key factor for VL-associated hemophagocytosis rather
spleen of L. donovani-infected mice, hemophagocytosis was than phenotypic changes on erythrocytes (Fig. 3). Further
most prominent in heavily infected macrophages, providing analyses by utilizing experimental models of VL will ad-
an argument against the idea that damages/changes to eryth- vance our understanding of pathologies in VL, especially
rocytes contribute significantly to this phenomenon. In fact, anemia, hopefully leading to development of drugs for
osmotic fragility of erythrocytes from the infected mice was symptom relief.

Fig. 3 Possible mechanisms of anemia during VL. Based on clinical contrast, L. donovani-infected mice present anemia along with
findings, possible mechanisms of anemia in VL include dysfunction in hemophagocytosis in the spleen, without showing signs of dysfunction
erythropoiesis due to myelofibrosis or iron deficiency, blood loss by in erythropoiesis, bleeding, anti-RBC antibody, or damage to RBC. A
bleeding, up-regulated RBC destruction by anti-RBC antibody, damage macrophage infected with amastigotes (arrowhead), phagocytosing
to RBC, or hemophagocytosis by hyper-activated macrophages. In RBCs (arrows), found in the spleen of a L. donovani-infected mouse
 Parasitol Res

Acknowledgments This work was supported in part by a grant from Cascio A, Colomba C, Antinori S, Orobello M, Paterson D, Titone L
the Takeda Science Foundation (http://www.takeda-sci.or.jp/index.html) (2002) Pediatric visceral leishmaniasis in Western Sicily, Italy: a
to YG. JC was involved in writing this review as a part of activities on the retrospective analysis of 111 cases. Eur J Clin Microbiol Infect Dis
UTokyo Amgen Scholars Program supported by Amgen Foundation. 21:277–282. doi:10.1007/s10096-002-0707-3
Castagnola E et al (1996) Early efficacy of liposomal amphotericin B in
Compliance with ethical standards the treatment of visceral leishmaniasis. Trans R Soc Trop Med Hyg
90:317–318
Conflict of interest The authors declare that they have no competing Chaliasos N, Bourantas C, Kritikou E, Lapatsanis P (1996) A 7-month-
interests. old girl with fever and bleeding. Lancet 347:1086
Chandra H, Chandra S, Kaushik RM (2013) Visceral leishmaniasis with
associated common, uncommon, and atypical morphological fea-
tures on bone marrow aspirate cytology in nonendemic region. J
Trop Med 2013:861032. doi:10.1155/2013/861032
Chang CS, Chang KP (1985) Heme requirement and acquisition by ex-
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