International Journal of Infectious Diseases
Volume 14, Issue 8 , Pages e682-e687, August 2010

Guanosine triphosphatases as novel therapeutic targets in tuberculosis

Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India

Received 8 May 2009; received in revised form 4 November 2009; accepted 9 November 2009. published online 08 March 2010.

Corresponding Editor: Sunit K. Singh, Hyderabad, India

Article Outline

Abstract 

Tuberculosis (TB) is an infectious disease caused by the aerobic microbe Mycobacterium tuberculosis H37Rv. Despite the availability of the Bacille Calmette–Guérin (BCG) vaccine and directly observed treatment, short-course (DOTS), TB is a leading cause of death and affects a third of the world's population. The most important factor associated with disease severity is the development of antibiotic-resistant strains, including multidrug-resistant (MDR)-TB and extensively drug-resistant (XDR)-TB. In order to understand disease pathogenesis, it is necessary to delineate the specific features of M. tuberculosis that enable it to evade the host defense system and contribute to its virulence. Here, we have reviewed the various characteristics, such as cell wall components, virulence genes, and the role of small guanosine triphosphatases (GTPases) in the pathogenesis of TB. GTPases are known to play a crucial role in the survival and pathogenesis of various pathogens. The key role of these proteins involves interference in phagosome maturation arrest, enabling pathogens to survive by escaping from lysozymes and toxic free radicals. This observation provides a new avenue for the development of anti-TB drugs.

Keywords: Mycobacterium, GTPase, Tuberculosis, Prokaryotes, Virulence factor, Signaling transduction

 

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1. Introduction 

Tuberculosis (TB) is a leading cause of mortality and affects one third of the world's population. This is a common and most deadly infectious disease, which spreads through the air when an affected person coughs, sneezes, or spits. It mainly attacks the lungs, but can also affect the central nervous system, lymphatic system, circulatory system, genitourinary system, gastrointestinal system, bones, joints, and skin.1 A number of treatment and preventive strategies have been implemented over the last 50 years, with the Bacille Calmette–Guérin (BCG) vaccine being the most widely used against TB. Some antibiotics, such as rifampin and isoniazid, are also employed in the treatment of TB. However, spontaneous mutation, incomplete and inadequate treatment, poor administrative control, and irregular distribution of drugs have led to the development of multidrug-resistant (MDR)-TB, progressing to extensively drug-resistant (XDR)-TB.2

Mycobacterium tuberculosis H37Rv is a unique acid-fast Gram-positive bacterium; it neither contains a phospholipid outer membrane nor retains dye due to the high lipid and mycolic acid content of its cell wall. The M. tuberculosis H37Rv cell wall contains large amounts of glycolipid and is especially rich in mycolic acid, peptidoglycan, lipoarabinomannan (LAM), phosphatidylinositol mannosides (PIM), phthiocerol dimycocerosate, cord factor, sulfolipids, and wax D (Figure 1).3, 4, 5, 6, 7

Several approaches executed from time to time have identified a number of genes conferring survival and persistence within the host. It manipulates host defense pathways resulting in inhibition of apoptosis of infected host cells. These virulence genes include nuoG, erp, phospholipases c, and fadE28.8, 9, 10, 11, 12, 13, 14

In the past few years, extensive work has been done to understand the role of guanosine triphosphatases (GTPases) in the growth and development of bacteria. GTPases are also known as molecular switch proteins.15 These proteins specifically bind and hydrolyze GTP, which in turn activates or inactivates the GTPase in a cyclic manner (Figure 2).15 GTPases are highly conserved and function through RNA or ribosome binding. G1, G2, G3, and G4 motifs are responsible for specific interactions with the guanine nucleotide and effector proteins. Genome sequencing projects have revealed a core of 11 universally conserved GTPases, referred to as elongation factors G and Tu (EF-G and EF-Tu), initiation factor 2 (IF2), LepA, Era, Obg, ThdF/TrmE, Ffh, FtsY, EngA, and YchF.15 These 11 GTPases are vital for bacterial life, since they regulate the cell cycle and distribution of DNA to daughter cells.16, 17, 18, 19

Eukaryotic Rab GTPases are important regulators of different steps of the pathways leading to phagosome maturation arrest. Rab GTPases, in particular Rab5 and Rab7, regulate rate-limiting steps leading to maintenance and self-preservation of Mycobacterium within host macrophages (Figure 3).20 Apart from these, Rab14 has also been identified as an important factor in maintaining the phagosome maturation block.21 Another GTPase, FtsZ, has been characterized as an assembling GTPase, which functions in prokaryotic cell division. FtsZ antagonists disrupt its assembling activity and cause lethality to a variety of bacteria.22 These studies show a crucial role of GTPases in the survival of Mycobacterium within the host macrophage.

  • View full-size image.
  • Figure 3. 

    Role of Rab GTPases in phagosome maturation arrest: recruitment of Rab5 on the phagosome results in the formation of early endosome, but lack of Rab7 blocks its maturation into late endosome, ultimately leading to the inhibition of phagosome lysosome fusion.

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2. Tuberculosis as the major problem we are facing today 

TB remains a global puzzle in spite of effective chemotherapy, the BCG vaccine, and the directly observed treatment, short-course (DOTS) strategy for its treatment. TB is a leading cause of mortality and morbidity and affects a third of the world's current population.2 Other Mycobacterium species such as Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, and Mycobacterium microti also cause TB, but are relatively less common. Latent infection is the most common and significant stage, which if left untreated leads to death in more than half of the patients.2 The BCG vaccine is one of the world's most widely used vaccines, despite showing variable effectiveness in different clinical trials. Treatment of TB employs anti-TB drugs to kill the bacteria, such as rifampin and isoniazid. A long time duration (around 6–12 months) is required to entirely eradicate Mycobacterium from the body.23 There are two stages in the infection process: latent TB and active TB, which demand different treatments. Latent TB is treated using a single drug, while active TB is best treated using a combination of several drugs, to reduce the development of antibiotic-resistant bacteria.24

Spontaneous mutations in M. tuberculosis H37Rv, incomplete and inadequate treatment,25, 26 poor administrative control on purchase, irregular distribution of the drugs, and improper quality control and bioavailability tests have led to the development of MDR-TB.27 MDR-TB is resistant to rifampicin and isoniazid.27 The DOTS strategy is a key factor involved in TB control.28 This strategy is based upon clinical trials done in the 1970s by the TB Research Center, Chennai, India. Mismanagement and ignorance in relation to treatment have led to the emergence of a new form of drug-resistant TB known as XDR-TB.29 Based on the meeting of the World Health Organization (WHO) XDR-TB task force, XDR-TB has been defined as TB caused by M. tuberculosis H37Rv resistant to at least rifampicin and isoniazid among the first-line anti-TB drugs (MDR-TB), as well as resistance to any fluoroquinolones, i.e., ofloxacin, ciprofloxacin, and levofloxacin, and at least one of three second-line anti-TB drugs, i.e., amikacin, kanamycin, and capreomycin.30 The increasing antibiotic resistance of M. tuberculosis H37Rv demands an urgent solution.

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3. Mycobacterium tuberculosis—natural warriors 

3.1. Unique cell wall structure 

The cell wall structure of M. tuberculosis H37Rv has become cynosure in the field of research, as it is unique among prokaryotes and is a major determinant of virulence for this bacterium. M. tuberculosis H37Rv contains large amounts of glycolipid and is especially rich in mycolic acid, making up approximately 60% of the cell wall. The orientation of mycolic acids is perpendicular to the plane of the membrane, creating a special lipid barrier, which serves as the major factor contributing to the virulence of M. tuberculosis H37Rv.3 In addition to mycolic acid, other glycolipids include LAM, PIM, phthiocerol dimycocerosate, cord factor/dimycolyltrehalose, sulfolipids, and wax D.4 Mycolic acids prevent an attack on Mycobacterium by cationic proteins, lysozymes, and the oxygen radicals in the phagocytic granule. Cord factor is toxic to mammalian cells and is also an inhibitor of polymorphonuclear neutrophil (PMN) migration, an important effector mechanism in the defense against external antigenic agents; cord factor is produced abundantly in virulent strains of M. tuberculosis H37Rv. Wax D in the cell envelope is the major component of Freund's complete adjuvant (CFA).

Antigen 85 complex is one of the dominant exported proteins and the most powerful protective antigen of M. tuberculosis H37Rv. It is a triad of related gene products. Antigen 85 is involved in disease pathogenesis through its fibronectin binding capacity. These proteins act as mycolyltransferases and are involved in the final stages of mycobacterial cell wall assembly.5

LAM is also an important component of the cell wall and has been shown to be involved in phagocytosis of M. tuberculosis H37Rv when ‘capped’ with short mannose oligosaccharides (ManLAM).4, 5 ManLAM specifically inhibits the pathway dependent on phosphatidylinositol-3-kinase (PI3K) and phosphatidylinositol-3-phosphate (PI3P) binding effectors, and in this way inhibits phagosome maturation (Figure 1).6 Porins in the mycobacterial cell wall show strikingly different features from those of other bacteria and form a sort of diffusion barrier that is 100–1000 times less permeable to hydrophilic molecules than that of other bacteria like Escherichia coli.7

3.2. Virulence factors 

M. tuberculosis H37Rv has an inbuilt capacity to survive and persist within a hostile environment and adverse conditions created by the host defense system. It is able to manipulate different host defense pathways and has the ability to actively inhibit the killing of infected host cells by apoptosis.8 This inhibition is caused by multiple genetic loci in M. tuberculosis H37Rv. It has been observed that the anti-apoptosis activity is attributable to the type 1 nicotinamide adenine dinucleotide (NADH) dehydrogenase of M. tuberculosis H37Rv, and is mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. Experiments in the SCID (severe combined immunodeficiency) mouse model have shown that the expression of M. tuberculosis H37Rv nuoG in non-pathogenic mycobacteria endowed them with the ability to inhibit apoptosis of infected macrophages, and increased their virulence. Deletion of nuoG in M. tuberculosis H37Rv destroyed its ability to inhibit macrophage apoptosis resulting in reduced virulence. This distinctly shows the straightforward link between the nuoG virulence factor and inhibition of macrophage apoptosis.8

Virulence also depends on the ability of Mycobacterium to multiply in mammalian hosts. The erp gene encodes an exported repetitive protein that causes multiplication of M. tuberculosis H37Rv in cultured macrophages.9

Parasitism of host macrophages by Mycobacterium is also a key factor in inducing virulence. Macrophage infection by Mycobacterium is a schematic process involving adhesions and phagocytosis-mediated entry into the host cell, ultimately leading to inhibition of phagosome–lysosome fusion and resistance to free radicals. Several approaches have been used from time to time to identify genes involved in virulence. One such approach is signature-tagged transposon mutagenesis, which has shown that mutants disrupted in the ATP-binding cassette (ABC) transporter-encoding genes Rv0986 and Rv0987 are impaired in their ability to bind to the host cell.10 An mRNA differential display assay has revealed distinctions between gene expression in M. tuberculosis H37Rv and its avirulent mutant strain H37Ra. It was found that six cDNAs that were expressed in H37Rv were not expressed in H37Ra; these were cloned and 10 inserts for each cDNA were sequenced, out of which six inserts were highly homologous to the M. tuberculosis H37Rv gene. Three of these genes, Rv2770c, Rv1345, and Rv0288, code for members of the PPE (Pro–Pro–Glu) protein family, a plausible polyketide synthase, and a member of the protein family containing ESAT-6 (early secretory antigenic target). These genes have been shown to be associated with pathogenesis of M. tuberculosis H37Rv.11

M. tuberculosis H37Rv uses inositol as a precursor in the production of its major thiol and lipoglycans. A mutant Mycobacterium lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyzes the first step in inositol synthesis, is viable only in the presence of high levels of exogenous inositol.12 M. tuberculosis H37Rv possesses four genes encoding phospholipases C, plcA, plcB, plcC, and plcD. Expression of these genes is up-regulated during the first 24h of macrophage infection.13 M. tuberculosis H37Rv uses fatty acids to persist inside the phagosome of macrophages. The fadE28 gene was identified as a source of fatty acids inside phagosomes, since the up-regulation of the fadE28 gene causes an elevation in fatty acids by increasing beta-oxidation.14

3.3. Dormant nature of MTB 

M. tuberculosis H37Rv latent bacilli are microorganisms that are well known for their ability to tolerate the stressful conditions produced by the immune system of the infected host against them. M. tuberculosis H37Rv counterbalances the adverse conditions and appears ‘silent’ to the immune system by slowing its metabolism or becoming dormant.31 The dormant bacilli are a major problem for TB treatment. Evidence from a murine model of TB has shown that nitric oxide synthase is essential for the infection. It has also been observed that oxygen and low, nontoxic concentrations of nitric oxide (NO) competitively modulate the expression of a 48-gene regulon, which is expressed in order to prepare bacilli for persistence during long periods of dormancy. NO has been found to be responsible for the reversible inhibition of aerobic respiration and growth. This inhibition of respiration by NO production and oxygen limitation within granulomas, suppress M. tuberculosis H37Rv replication rates in individuals with latent TB.32 Seminal studies have shown that gradual depletion in oxygen concentrations causes a non-replicating persistent stage characterized by bacteriostasis and metabolic, chromosomal, and structural changes in the dormant bacteria. A further reduction in oxygen tension produces a more quiescent state, characterized by the onset of sensitivity to metronidazole and resistance to other antimicrobials. Studies have been focused on NO as an immune factor that is able to suppress mycobacterial replication rates in vivo and is involved in the initiation and maintenance of the latent state in conjunction with low tissue oxygen concentrations.33

3.4. GTPases as versatile signaling molecules 

GTPases are regarded as molecular switch proteins based on their special mode of action. Each protein is capable of selectively binding and hydrolyzing guanosine triphosphate (GTP) in a cyclic manner that activates and inactivates the GTPase protein (Figure 2).15 These GTPases comprise a protein superfamily of highly conserved molecular switches capable of performing a diverse range of functions. GTPases make a significant contribution to different fundamental cellular processes, which vary between prokaryotes and eukaryotes. In contrast to prokaryotes, eukaryotic G-proteins, commonly known as heterotrimeric G-proteins, consist of three subunits – α, β, and γ. The G-domain is located on the largest, i.e., α-subunit, forming a tightly associated protein complex with the two other smaller subunits β and γ. G-protein coupled receptors act as the specific reaction partners of heterotrimeric G-proteins. In the normal state the GTPase is inactive, but upon receptor activation, the GTPase is activated by the intracellular receptor domain, which in turn activates other steps of the signal transduction pathway.16 One striking difference between GTPases of prokaryotes and eukaryotes is that prokaryotes do not use GTPases in the same way as eukaryotes to regulate membrane signaling.19

Investigations and studies carried out to understand the role of GTPases have revealed an important conserved feature of these proteins, i.e., they implement their function through interaction with RNA or ribosomes. The GTPase passes through three conformational states: initially the GTPase is not bound to any nucleotide and is inactive; after binding to GTP it becomes active; and after interaction with the target GTP it is hydrolyzed, resulting in the inactivation of GTPase, with GDP bound to it. The empty (inactivated) state is regenerated by interaction with another factor that catalyses nucleotide release.19 Studies have recognized G1, G2, G3, and G4 sequence motifs as the highly conserved elements that are involved in specific interactions with the guanine nucleotides and effector proteins. Of these motifs, G1, G3, and G4 have been found to bind and hydrolyze GTP and also interact with cofactor Mg2+.15 The G2 motif is known as the effector domain, which undergoes a conformational change essential for GTPase function.34, 35 This particular motif is characteristic for each subfamily of GTPases, since it is not conserved throughout the GTPase superfamily. Bacterial GTPases are the principal regulators of ribosome function and the distribution of DNA to daughter cells following cell division.

Genome sequencing projects have demonstrated that bacteria possess a core of 11 universally conserved GTPases, named EF-G and EF-Tu, IF2, LepA, Era, Obg, ThdF/TrmE, Ffh, FtsY, EngA, and YchF.36 Among all these GTPases, EF-G, EF-Tu, and IF2 are multidomain GTPases with essential roles in the initiation and elongation phases of translation. They bind to the site on the ribosomes where their low intrinsic GTPase activities are strongly induced. IF2, EF-G, EF-Tu, and RF3 are the four proteins belonging to the GTPase superfamily that participate in bacterial biosynthesis.17, 18 Obg was discovered in Bacillus subtilis and has been implicated in regulating sporulation, chromosome partitioning, mycelium development, and the stress response. Studies have shown that it is also involved in the initiation of chromosome replication37 and the regulation of the phosphorylation state of SpoOA, resulting in the initiation of sporulation. It promotes binding of tRNA to the ribosomal P site during the initiation step of translation.38, 39 EF-Tu is involved in the delivery of an amino acyl-tRNA to the A site and the formation of the peptide bond. EF-G promotes translocation of the ribosome.40, 41 LepA is universally conserved among all bacterial GTPases.19, 42 Era was first described in E. coli, and it has been postulated that the B. subtilis Era ortholog functions in chromosome segregation.43, 44 The 16s RNA-bound form of Era is an activator of cell division and the RNA free form is an inactive state required for the termination of cell division. ThdF/TrmE is an Era-like GTPase with a GTP binding domain and is essential for the normal function of the protein synthesis apparatus.19, 45 EngA is a unique GTPase as it contains two GTP binding domains arranged in tandem.36 It is thought to be involved in ribosome assembly or stability. A myriad of experiments and studies have been conducted on these GTPases from time to time, and it has been observed that in all bacterial systems the core 11 GTPases are vital for life and for regulating ribosome function directly or indirectly by transmitting signals, regulating the cell cycle, DNA partitioning, and DNA segregation.19

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4. GTPases as an attractive target 

M. tuberculosis H37Rv, like other intracellular bacterial pathogens, has evolved highly specialized mechanisms to enter and survive intracellularly within host macrophages. M. tuberculosis H37Rv inhibits phagosome maturation and so remains protected from degradative and bactericidal intracellular compartments, lysozymes, and antigen-presenting organelles in the host phagocytic cells.20 Therefore phagosome maturation arrest is the central power of M. tuberculosis H37Rv and the cause of loopholes used by M. tuberculosis H37Rv against the host defense system.

Studies have shown that GTPases play a central role in phagosome maturation arrest as described in a study highlighting the role of Rab GTPases.46 GTP binding proteins of the Rab family have been observed to regulate transport in the endocytic and exocytic pathways and in conferring specificity of vesicle fusion. Studies have shown that inhibition of phagosome maturation is due to a block between the maturation phases controlled by the early endocytic small GTPase Rab5 and the late endosomal small GTPase Rab7 (Figure 3).46 Transport from the plasma membrane to early endosomes and the homotypic fusion of early endosomes is controlled by Rab5. Rab4 is involved in trafficking from the early endosomes through the recycling pathway, whereas transport from the early to late endosomes is controlled by Rab7. Rab5 is known as a regulator of a rate-limiting step in early endosomal fusion and this is responsible for the maintenance and self-preservation of the early endosomal sorting compartment. Mycobacteria are able to survive and multiply inside the macrophage due to the lack of recruitment of Rab7.46

Recently it has been observed that phagosome maturation arrest is due to a block in the PI3K-dependent trafficking pathway from the trans-Golgi network (TGN) to the phagosome.6 PI3K is central to cell survival and protects cells from programmed cell death and is important for regulating neutrophil function and inflammatory processes. This block prevents the delivery of the critical late endosomal and lysosomal effectors, such as V0-H+-ATPase subunits, and lysosomal hydrolases, such as cathepsin D, to the M. tuberculosis H37Rv phagosome.6

PI3P has been shown to play an essential role in several signaling pathways and endocytic traffic, and is generated on the early endosomes via recruitment of PI3Ks, including hVPS34, by the active Rab5. Two bacterial products produced by M. tuberculosis H37Rv act on PI3P to inhibit phagosome maturation: (1) LAM, the well known unique cell wall component of M. tuberculosis H37Rv, prevents PI3P generation on phagosomes,6 and (2) the bacterial enzyme SapM acts as a PI3P phosphatase to remove this lipid from phagosomes.47 Hence Mycobacterium effectively blocks their delivery to lysosomes by inhibiting PI3P action.

The mechanism behind modification of Rabs associated with phagosome maturation has been suggested. Rab22a, a member of the group 5 Rabs, has been reported to accumulate on mycobacterial phagosomes. It has also been shown to be responsible for phagosome maturation arrest in an experiment that demonstrated that reduced expression of this GTPase enhanced phagosome maturation in phagosomes with live mycobacteria, while over-expression of its mutant prevented phagosome maturation. From this study it was concluded that Rab22a is noteworthy for the regulation of Rab7 conversions on phagosomes and so in phagosome maturation.21

The role of another GTPase, Rab14, has been shown by Kyei et al., who observed the accumulation of Rab14 on phagosomes harboring live bacteria as demonstrated by 4-D microscopy.22 This study also reported the evaluative role of Rab14 in maintaining the phagosome maturation block. They considered that the recruitment of Rab14 as a knockdown by SiRNA or over-expression of Rab14 dominant negative mutants (Rab14S25N and Rab140N125I) relinquished the maturation block and caused phagosome maturation into phagolysosomes. These findings suggest that M. tuberculosis target Rab GTPase functions to colonize and persist within phagosomes and remain protected from the host defense system, hence Rab GTPases serve as potential drug targets against M. tuberculosis H37Rv.

Apart from Rabs, another GTPase has also been reported – FtsZ; this is an assembling GTPase that is a prokaryotic cell division protein.48 FtsZ assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. FtsZ functions in a manner similar to eukaryotic tubulin and polymerizes into dynamic protofilaments in the presence of GTP accompanied by GTP hydrolysis after polymer assembly, as well as directing the formation of the septosome between daughter cells. Margalit et al. identified zantrins as compounds that inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or inducing filament hyper-stability through increased lateral association; in this way they disrupt FtsZ ring assembly in E. coli and cause lethality to a variety of bacteria in broth cultures.48 This clearly shows that FtsZ antagonists can potentially serve as a source for the development of new broad-spectrum antibacterial drugs, also able to target M. tuberculosis H37Rv. It is an attractive and novel therapeutic target for the development of new antibiotics.49

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5. Conclusions 

The development of anti-TB drugs has been the topic of discussion since the introduction of BCG and DOTS. As the rate of development of new drugs has increased, antibiotic resistance in the strain has also increased, leading to the emergence of MDR-TB and XDR-TB. This increasing antibiotic resistance demands a novel way to design drugs active against TB. Signaling molecule GTPases such as Era, Obg, LepA, and FtsZ are vital for growth and development and for specific cellular functions in the bacteria. In view of all the observations that have shown a crucial role for GTPases in cell division, the cell cycle, bacterial biosynthesis, gene translation, distribution of DNA to daughter cells, and sporulation, it is concluded that a more comprehensive approach is needed to design a new generation of drugs for TB using GTPases. The most important factor contributing to the ability of the pathogen to survive host inflammatory mediators and antibiotic treatment is its dormant state.

5.1. Future perspectives 

The establishment of the latent state requires unique regulatory pathways and GTPases, as shown by eukaryotic Rab GTPases in the survival and persistence of M. tuberculosis within host macrophages. This highlights the possible use of GTPases as novel drug targets against TB. Previous studies have shown an important role of GTP-binding proteins in ribosome assembly and DNA segregation. GTP binding proteins can be deleted in order to understand their function. This study serves as a basis to understand the ribosome-associated function of Era, Obg, LepA, EngA, and other GTPases in M. tuberculosis through polysome profiling. It will require a huge effort to realize the significance of these GTPases in the physiology of M. tuberculosis and to design an effective drug against the recalcitrant form of M. tuberculosis.

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Acknowledgements 

We thank Dr Rajesh S. Gokhale for making this work possible. We also thank Dr Hemant Khanna (University of Michigan, USA) for valuable suggestions. The authors acknowledge financial support from GAP0050 of the DST (Department of Science and Technology, Government of India) and CSIR (Council of Scientific and Industrial Research).

Conflict of interest: No conflict of interest to declare.

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PII: S1201-9712(10)01465-7

doi:10.1016/j.ijid.2009.11.016

International Journal of Infectious Diseases
Volume 14, Issue 8 , Pages e682-e687, August 2010

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