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    ABSTRACT: Early detection and confirmation of cholera outbreaks are crucial for rapid implementation of control measures. Because cholera frequently affects regions with limited laboratory resources, rapid diagnostic tests (RDT) designed for field conditions are important to enhance rapid response. Stool culture remains the "gold standard" for cholera diagnosis; however, its lack of sensitivity may lead to underestimation of test specificity. We evaluated the Crystal VC® immunochromatographic test (Span Diagnostics, India) for cholera diagnosis using a modified reference standard that combines culture-dependent and independent assays, or a Bayesian latent class model (LCM) analysis. The study was conducted during a cholera epidemic in 2008, in Lubumbashi, Democratic Republic of Congo. Stools collected from 296 patients were used to perform the RDT on site and sent to Institut Pasteur, Paris, for bacterial culture. In comparison with culture as the gold standard, the RDT showed good sensitivity (92.2%; 95% CI: 86.8%-95.9%) but poor specificity when used by a trained laboratory technician (70.6%; 95% CI: 60.7%-79.2%) or by clinicians with no specific test training (60.4%, 95% CI: 50.2%-70.0%). The specificity of the test performed by the laboratory technician increased to 88.6% (95% CI: 78.7-94.9) when PCR was combined with culture results as the reference standard, and to 85.0% (95% CI: 70.4-99.2), when the Bayesian LCM analysis was used for performance evaluation. In both cases, the sensitivity remained high. Using an improved reference standard or appropriate statistical methods for diagnostic test evaluations in the absence of a gold standard, we report better performance of the Crystal VC® RDT than previously published. Our results confirm that this test can be used for early outbreak detection or epidemiological surveillance, key components of efficient global cholera control. Our analysis also highlights the importance of improving evaluations of RDT when no reliable gold standard is available.
    PLoS ONE 05/2012; 7(5):e37360. DOI:10.1371/journal.pone.0037360 · 3.53 Impact Factor
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    ABSTRACT: During two surveys conducted in 2008 and 2009, the culture method described in the international standard ISO/TS 21872-1 was applied to the detection of Vibrio parahaemolyticus and Vibrio cholerae in 112 living bivalve mollusc samples, with a chromogenic medium used in addition to the TCBS agar, as second selective isolation medium and for enumeration of V. parahaemolyticus and V. cholerae by surface inoculation. A PCR method for detection of these 2 Vibrio species and the hemolysin genes tdh and trh, was applied in parallel. In 2009, the survey was extended to finfish fillets and crustaceans. PCR was also used for species confirmation of characteristic colonies. The identity of the PCR products, specifically targeting V. parahaemolyticus, was checked by sequencing. Occurrence of V. parahaemolyticus and V. cholerae isolates in living bivalve molluscs ranged from 30.4% to 32.6% and from 1.4% to 4.7% respectively. In frozen crustaceans (2009 survey) V. parahaemolyticus and V. cholerae isolates were respectively found in 45% and 10% of the samples. No V. parahaemolyticus or V. cholerae was detected in frozen fish fillets, neither by the ISO method nor by PCR. In 2009, enteropathogenic V. parahaemolyticus (trh+) was isolated from 4 out of 43 oyster samples while the trh gene was present in V. alginolyticus strains and in samples where V. parahaemolyticus was not detected (9 over 112 samples). The ISO method failed to isolate V. parahaemolyticus in 44% to 53% of the living bivalve molluscs where PCR detected the toxR gene specific of V. parahaemolyticus (Vp-toxR). Our results highlighted the need for a revision of the ISO/TS 21872-1 standard, at least, for analysis of living bivalve molluscs, and confirmed the increasing concern of enteropathogenic V. parahaemolyticus in French bivalve molluscs. Enrichment at 41.5°C was questioned and some reliable solutions for the improvement of the ISO/TS 21872-1 method, such as the PCR method for screening of positive samples and confirmation of colonies, were pointed out.
    International journal of food microbiology 05/2012; 157(2):189-94. DOI:10.1016/j.ijfoodmicro.2012.04.026 · 3.16 Impact Factor
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    ABSTRACT: The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R) genes. It was shown that evolution of M. tuberculosis complex strains can be studied using 3R SNPs and a high-resolution tool for strain discrimination was developed. Here we investigated the genetic diversity and propose a phylogeny for Beijing strains by analyzing polymorphisms in 3R genes. A group of 3R genes was sequenced in a collection of Beijing strains from different geographic origins. Sequence analysis and comparison with the ones of non-Beijing strains identified several SNPs. These SNPs were used to type a larger collection of Beijing strains and allowed identification of 26 different sequence types for which a phylogeny was constructed. Phylogenetic relationships established by sequence types were in agreement with evolutionary pathways suggested by other genetic markers, such as Large Sequence Polymorphisms (LSPs). A recent Beijing genotype (Bmyc10), which included 60% of strains from distinct parts of the world, appeared to be predominant. We found SNPs in 3R genes associated with the Beijing family, which enabled discrimination of different groups and the proposal of a phylogeny. The Beijing family can be divided into different groups characterized by particular genetic polymorphisms that may reflect pathogenic features. These SNPs are new, potential genetic markers that may contribute to better understand the success of the Beijing family.
    PLoS ONE 01/2011; 6(1):e16020. DOI:10.1371/journal.pone.0016020 · 3.53 Impact Factor
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    ABSTRACT: The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.
    PLoS Pathogens 09/2010; 6(9):e1001100. DOI:10.1371/journal.ppat.1001100 · 8.06 Impact Factor
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    ABSTRACT: Multidrug-resistant (MDR) strains were identified in 40% of 54 strains from patients presenting with tuberculosis (TB) treatment failure or relapse in Bangui, Central African Republic. Results obtained with the MTBDRplus line-probe assay or rpoB sequencing were 86% concordant with rifampicin (RMP) resistant phenotypes, while the amplification refractory mutation system test was 71% concordant. No mutation was found in RMP-susceptible strains. MTBDRplus and sequencing were concordant with the detection of the S315T mutation in katG in 95% of MDR strains. Sequencing of pncA suggested pyrazinamide resistance in 50% of MDR strains. Knowledge of these resistances should help to implement treatment in low-income countries.
    The International Journal of Tuberculosis and Lung Disease 06/2010; 14(6):782-5. · 2.76 Impact Factor
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    ABSTRACT: Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task. We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains. This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.
    PLoS ONE 02/2008; 3(2):e1538. DOI:10.1371/journal.pone.0001538 · 3.53 Impact Factor
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    ABSTRACT: Isoxyl (ISO), a thiourea derivative that was successfully used for the clinical treatment of tuberculosis during the 1960s, is an inhibitor of the synthesis of oleic and mycolic acids in Mycobacterium tuberculosis. Its effect on oleic acid synthesis has been shown to be attributable to its inhibitory activity on the stearoyl-coenzyme A desaturase DesA3, but its enzymatic target(s) in the mycolic acid pathway remains to be identified. With the goal of elucidating the mode of action of ISO, we have isolated a number of spontaneous ISO-resistant mutants of M. tuberculosis and undertaken their genotypic characterization. We report here the characterization of a subset of these strains carrying mutations in the monooxygenase gene ethA. Through complementation studies, we demonstrate for the first time that the EthA-mediated oxidation of ISO is absolutely required for this prodrug to inhibit its lethal enzymatic target(s) in M. tuberculosis. An analysis of the metabolites resulting from the in vitro transformation of ISO by purified EthA revealed the occurrence of a formimidamide allowing the formulation of an activation pathway in which the oxidation of ISO catalyzed by EthA is followed by chemical transformations involving extrusion or elimination and, finally, hydrolysis.
    Antimicrobial Agents and Chemotherapy 12/2007; 51(11):3824-9. DOI:10.1128/AAC.00433-07 · 4.45 Impact Factor
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    ABSTRACT: The contribution of horizontal gene transfer (HGT) to the evolution of Mycobacterium tuberculosis -- the main causal agent of tuberculosis in humans -- and closely related members of the M. tuberculosis complex remains poorly understood. Using a combination of genome-wide parametric analyses, we have identified 48 M. tuberculosis chromosomal regions with atypical characteristics, potentially due to HGT. These specific regions account for 4.5% of the genome (199 kb) and include 256 genes. Many display features typical of the genomic islands found in other bacteria, including residual material from mobile genetic elements, flanking direct repeats, insertion in the vicinity of tRNA sequences, and genes with putative or documented virulence functions. Southern blotting analysis of nine of these 48 regions confirmed their presence in "Mycobacterium prototuberculosis," the ancestral species of the M. tuberculosis complex. Finally, our results strongly suggest that the ancestor of the tubercle bacilli was an environmental bacillus that exchanged genetic material with other bacterial species, including Proteobacteria in particular, present in its surroundings. This study describes a rational approach to searching for mycobacterial virulence genes, and highlights the importance of dissecting gene transfer networks to improve our understanding of mycobacterial pathogenicity and evolution.
    Molecular Biology and Evolution 09/2007; 24(8):1861-71. DOI:10.1093/molbev/msm111 · 14.31 Impact Factor
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    ABSTRACT: Using signature-tagged transposon mutagenesis, we isolated 23 Mycobacterium tuberculosis mutants, corresponding to 21 genes or genetic regions, attenuated in their ability to parasitize human macrophages. Mutants disrupted in the ABC transporter-encoding genes Rv0986 and Rv0987 were further characterized as being impaired in their ability to bind to host cells.
    Infection and Immunity 02/2007; 75(1):504-7. DOI:10.1128/IAI.00058-06 · 4.16 Impact Factor
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    ABSTRACT: Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR). In this study, 55 MDR and 194 non-MDR strains were analyzed. Variations in DNA repair genes ada/alkA and ogt were identified. Among them, by comparison to M. tuberculosis published sequences, we found a non-sense variation in ada/alkA gene which was also observed in M. bovis AF2122 strain. SNPs that are present in the adjacent regions to the amber variation are different in M. bovis and in M. tuberculosis strain. An Amber codon was found in the ada/alkA locus of clustered M. tuberculosis isolates and in M. bovis strain AF2122. This is likely due to convergent evolution because SNP differences between strains are incompatible with horizontal transfer of an entire gene. This suggests that such a variation may confer a selective advantage and be implicated in hypermutator phenotype expression, which in turn contributes to adaptation to environmental changes.
    BMC Microbiology 02/2007; 7:39. DOI:10.1186/1471-2180-7-39 · 2.98 Impact Factor
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    ABSTRACT: We investigated multidrug-resistant (MDR) Mycobacterium tuberculosis strains in Bangui, Central African Republic. We found 39.6% with the same spoligotype and synonymous single nucleotide polymorphism in the mutT1 gene. However, strains had different rpoB mutations responsible for rifampin resistance. MDR strains in Bangui may emerge preferentially from a single, MDR-prone family.
    Emerging infectious diseases 10/2006; 12(9):1454-6. DOI:10.3201/eid1209.060361 · 7.33 Impact Factor
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    ABSTRACT: Cell envelope lipids play an important role in the pathogenicity of mycobacteria, but the mechanisms by which they are transported to the outer membrane of these prokaryotes are largely unknown. Here, we provide evidence that LppX is a lipoprotein required for the translocation of complex lipids, the phthiocerol dimycocerosates (DIM), to the outer membrane of Mycobacterium tuberculosis. Abolition of DIM transport following disruption of the lppX gene is accompanied by an important attenuation of the virulence of the tubercle bacillus. The crystal structure of LppX unveils an U-shaped beta-half-barrel dominated by a large hydrophobic cavity suitable to accommodate a single DIM molecule. LppX shares a similar fold with the periplasmic molecular chaperone LolA and the outer membrane lipoprotein LolB, which are involved in the localization of lipoproteins to the outer membrane of Gram-negative bacteria. Based on the structure and although an indirect participation of LppX in DIM transport cannot yet be ruled out, we propose LppX to be the first characterized member of a family of structurally related lipoproteins that carry lipophilic molecules across the mycobacterial cell envelope.
    The EMBO Journal 05/2006; 25(7):1436-44. DOI:10.1038/sj.emboj.7601048 · 10.75 Impact Factor
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    ABSTRACT: Mycobacterium tuberculosis and Mycobacterium smegmatis MutT1, MutT2, MutT3, and Rv3908 (MutT4) enzymes were screened for an antimutator role. Results indicate that both MutT1, in M. tuberculosis and M. smegmatis, and MutT4, in M. smegmatis, have that role. Furthermore, an 8-oxo-guanosine triphosphatase function for MutT1 and MutT2 is suggested.
    Journal of Bacteriology 05/2006; 188(8):3159-61. DOI:10.1128/JB.188.8.3159-3161.2006 · 2.69 Impact Factor
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    ABSTRACT: Multidrug-resistant tuberculosis was diagnosed in 21 HIV-negative, nonhospitalized male patients residing in northern Tunisia. A detailed investigation showed accelerated transmission of a Mycobacterium tuberculosis clone of the Haarlem type in 90% of all patients. This finding highlights the epidemic potential of this prevalent genotype.
    Emerging infectious diseases 07/2005; 11(6):957-61. DOI:10.3201/eid1106.041365 · 7.33 Impact Factor
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    ABSTRACT: Rv1395 is annotated as a potential transcriptional regulator of the AraC family. The Rv1395 insertional mutant was identified in a signature tag mutagenesis study in Mycobacterium tuberculosis and was shown to be attenuated in the lungs of mice. Here, we used comparative genomics and biochemical methods to show that Rv1395 is unique to the M. tuberculosis complex and that it encodes a protein that binds the region between two divergent genes, a member of the cytochrome P450 family (Rv1394c or cyp132) and Rv1395 itself. Rv1395 binds to this DNA region by its helix-turn-helix-containing C-terminal domain, and it recognizes two sites with different affinity. We identified the transcriptional start points (TSP) of Rv1394c and Rv1395: both genes have two TSPs, three of which are located in the intergenic region. We constructed and compared various transcriptional fusions consisting of the promoter regions and a reporter gene in Mycobacterium smegmatis: this showed that Rv1395 induces the expression of the cytochrome P450 gene (Rv1394c) and represses its own transcription. This was confirmed in M. tuberculosis when the wild type and a Rv1395-overexpressing strain were used as hosts for the fusions. Site-directed mutagenesis showed that Rv1395 binds to the two sites in a co-operative manner and that binding to both sites is required for Rv1395 optimal activity. A model describing the potential mode of action of Rv1395 is discussed.
    Journal of Biological Chemistry 10/2003; 278(36):33763-73. DOI:10.1074/jbc.M305963200 · 4.60 Impact Factor
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    ABSTRACT: Alterations in genes involved in the repair of DNA mutations (mut genes) result in an increased mutation frequency and better adaptability of the bacterium to stressful conditions. W-Beijing genotype strains displayed unique missense alterations in three putative mut genes, including two of the mutT type (Rv3908 and mutT2) and ogt. These polymorphisms were found to be characteristic and unique to W-Beijing phylogenetic lineage. Analysis of the mut genes in 55 representative W-Beijing isolates suggests a sequential acquisition of the mutations, elucidating a plausible pathway of the molecular evolution of this clonal family. The acquisition of mut genes may explain in part the ability of the isolates of W-Beijing type to rapidly adapt to their environment.
    Emerging infectious diseases 08/2003; 9(7):838-45. DOI:10.3201/eid0907.020589 · 7.33 Impact Factor
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    ABSTRACT: Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence. Here, we investigated this possibility by inactivating the M. tuberculosis Nramp1 gene (Mramp) by allelic exchange mutagenesis. Disruption of Mramp did not affect the extracellular growth of bacteria under standard conditions. However, the Mramp mutation was associated with growth impairment under conditions of limited iron availability. The Mramp mutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or in Nramp1(+/+) and Nramp1(-/-) congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental and Mramp mutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate that Mramp does not contribute to the virulence of M. tuberculosis in mice.
    Infection and Immunity 09/2002; 70(8):4124-31. DOI:10.1128/IAI.70.8.4124-4131.2002 · 4.16 Impact Factor
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    ABSTRACT: Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild-type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the Delta plcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT-PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.
    Molecular Microbiology 08/2002; 45(1):203-17. DOI:10.1046/j.1365-2958.2002.03009.x · 5.03 Impact Factor
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    ABSTRACT: Staphylococcus aureus nuclease is a small, secreted protein which has been successfully used as a reporter system to identify exported products in Lactococcus lactis. Here, biochemical evidence is provided that the nuclease is exported by Mycobacterium smegmatis in the presence, but also in the absence of a signal sequence, and thus probably independently of the Sec translocation pathway. This implies that the nuclease should not be used as a reporter system in mycobacteria for the identification of exported products, despite what has been reported previously in the literature. The nuclease can be extended to create hybrid proteins that remain compatible with its secretion, whereas some other shorter fusions are not tolerated. This suggests that correct folding is required for efficient export. Extensive mutational analysis did not identify a specific secretion pathway. This suggests that the nuclease may be exported by different redundant systems or that components of this alternative Sec pathway are essential for bacterial survival.
    Microbiology 03/2002; 148(Pt 2):529-36. · 2.84 Impact Factor
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    ABSTRACT: Erp (exported repeated protein) was originally characterized as a virulence factor in Mycobacterium tuberculosis and was thought to be present only in Mycobacterium leprae and members of the TB complex. Here it is shown that Erp is a ubiquitous extracellular protein found in all of the mycobacterial species tested. Erp proteins have a modular organization and contain three domains: a highly conserved amino-terminal domain which includes a signal sequence, a central variable region containing repeats based on the motif PGLTS, and a conserved carboxy-terminal domain rich in proline and alanine. The number and fidelity of PGLTS repeats of the central region differ considerably between mycobacterial species. This region is, however, identical in all of the clinical M. tuberculosis strains tested. In addition, it is shown here that a Mycobacterium smegmatis erp::aph mutant displays altered colony morphology which is complemented by all the Erp orthologues tested. The genome sequence flanking the erp gene includes cell-wall-related ORFs and displays extensive conservation between saprophytic and pathogenic mycobacteria.
    Microbiology 09/2001; 147(Pt 8):2315-20. · 2.84 Impact Factor

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