Publications by authors named "Viviana Ritacco"

62 Publications

SARS-CoV-2 variants and the so-called resistance to vaccines.

Medicina (B Aires) 2021 ;81(3):421-426

Comité de Redacción Medicina (B Aires), Buenos Aires, Argentina.

RNA viruses (except retroviruses) replicate by the action of an RNA-dependent RNA polymerase, which lacks a proofreading exonuclease and, consequently, errors may occur in each replication giving place to viral mutants. Depending on their fitness, these mutants either become extinct or thrive, spawning variants that escape the immune system. The most important SARS-CoV-2 mutations are those that alter the amino acid sequence in the viral S protein because this protein holds the key for the virus to enter the human cell. The more viruses replicate, the more they mutate, and the more likely it is that dominant resistant variants will appear. In such cases, more stringent measures for community protection will be required. Vaccines and polyclonal antibodies, which induce a response directed towards several sites along the S protein, would maintain effective protection against SARS-CoV-2 variants. Furthermore, vaccines appear to induce an increased helper and cytotoxic T-cell response, which may also be a biomarker of protection. In densely populated areas with insufficient protection measures, the virus spreads freely, thus increasing the likelihood of generating escape mutants. India and Manaus exemplify this situation. Natural evolution selects the mutants that multiply most efficiently without eliminating the host, thus facilitating their spread. Contrastingly, the circulation of viruses of high virulence and lethality (Ebola, hantavirus) that eliminate the host remain limited to certain geographic areas, without further dissemination. Therefore, it would be expected that SARS-CoV-2 will evolve into more infectious and less virulent variants.
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June 2021

Five-year microevolution of a multidrug-resistant Mycobacterium tuberculosis strain within a patient with inadequate compliance to treatment.

BMC Infect Dis 2021 Apr 29;21(1):394. Epub 2021 Apr 29.

Instituto Nacional de Enfermedades Infecciosas-ANLIS Carlos Malbrán, Buenos Aires, Argentina.

Background: Whole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution -the genetic variability of M. tuberculosis at short time scales- of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported.

Case Presentation: In this work, we applied whole genome sequencing analysis for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium tuberculosis isolates obtained from a patient within 57-months of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5' untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patient, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy.

Conclusions: This report highlights the relevance of whole-genome sequencing analysis in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.
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http://dx.doi.org/10.1186/s12879-021-06069-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082761PMC
April 2021

Tuberculosis and COVID-19: a dangerous relationship.

Medicina (B Aires) 2020 ;80 Suppl 6:117-118

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January 2021

Recurrences of multidrug-resistant tuberculosis: Strains involved, within-host diversity, and fine-tuned allocation of reinfections.

Transbound Emerg Dis 2021 Jan 7. Epub 2021 Jan 7.

Hospital General Universitario Gregorio Marañón, Madrid, Spain.

Recurrent tuberculosis occurs due to exogenous reinfection or reactivation/persistence. We analysed 90 sequential MDR Mtb isolates obtained in Argentina from 27 patients with previously diagnosed MDR-TB that recurred in 2018 (1-10 years, 2-10 isolates per patient). Three long-term predominant strains were responsible for 63% of all MDR-TB recurrences. Most of the remaining patients were infected by strains different from each other. Reactivation/persistence of the same strain caused all but one recurrence, which was due to a reinfection with a predominant strain. One of the prevalent strains showed marked stability in the recurrences, while in another strain higher SNP-based diversity was observed. Comparisons of intra- versus inter-patient SNP distances identified two possible reinfections with closely related variants circulating in the community. Our results show a complex scenario of MDR-TB infections in settings with predominant MDR Mtb strains.
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http://dx.doi.org/10.1111/tbed.13982DOI Listing
January 2021

[About population tests to detect anti-SARS-CoV-2 antibodies].

Medicina (B Aires) 2020 ;80 Suppl 3:87-88

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July 2020

Association between bacterial homoplastic variants and radiological pathology in tuberculosis.

Thorax 2020 07 16;75(7):584-591. Epub 2020 Jun 16.

UCL Genetics Institute, University College London, London, UK.

Background: Understanding how pathogen genetic factors contribute to pathology in TB could enable tailored treatments to the most pathogenic and infectious strains. New strategies are needed to control drug-resistant TB, which requires longer and costlier treatment. We hypothesised that the severity of radiological pathology on the chest radiograph in TB disease was associated with variants arising independently, multiple times (homoplasies) in the genome.

Methods: We performed whole genome sequencing (Illumina HiSeq2000 platform) on isolates from 103 patients with drug-resistant TB in Lima between 2010 and 2013. Variables including age, sex, HIV status, previous TB disease and the percentage of lung involvement on the pretreatment chest radiograph were collected from health posts of the national TB programme. Genomic variants were identified using standard pipelines.

Results: Two mutations were significantly associated with more widespread radiological pathology in a multivariable regression model controlling for confounding variables (Rv2828c.141, RR 1.3, 95% CI 1.21 to 1.39, p<0.01; rpoC.1040 95% CI 1.77 to 2.16, RR 1.9, p<0.01). The rpoB.450 mutation was associated with less extensive radiological pathology (RR 0.81, 95% CI 0.69 to 0.94, p=0.03), suggestive of a bacterial fitness cost for this mutation in vivo. Patients with a previous episode of TB disease and those between 10 and 30 years of age also had significantly increased radiological pathology.

Conclusions: This study is the first to compare the genome to radiological pathology on the chest radiograph. We identified two variants significantly positively associated with more widespread radiological pathology and one with reduced pathology. Prospective studies are warranted to determine whether mutations associated with increased pathology also predict the spread of drug-resistant TB.
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http://dx.doi.org/10.1136/thoraxjnl-2019-213281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361023PMC
July 2020

A Phenotypic Characterization of Two Isolates of a Multidrug-Resistant Outbreak Strain of with Opposite Epidemiological Fitness.

Biomed Res Int 2020 8;2020:4741237. Epub 2020 Apr 8.

Institute of Biotechnology, National Institute of Agricultural Technology (INTA)/IABIMO-CONICET (Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria), Argentina.

Tuberculosis (TB) is an infectious disease, caused by , primarily affecting the lungs. The strain of the Haarlem family named M was responsible for a large multidrug-resistant TB (MDR-TB) outbreak in Buenos Aires. This outbreak started in the early 1990s and in the mid 2000s still accounted for 29% of all MDR-TB cases in Argentina. By contrast, a clonal variant of strain M, named 410, has caused a single tuberculosis case since the onset of the outbreak. The molecular bases of the high epidemiological fitness of the M strain remain unclear. To assess its unique molecular properties, herein, we performed a comparative protein and lipid analysis of a representative clone of the M strain (Mp) and the nonprosperous M variant 410. We also evaluated their growth in low pH. The variant 410 had higher levels of latency proteins under standard conditions and delayed growth at low pH, suggesting that it is more sensitive to stress stimuli than Mp. Moreover, Mp showed higher levels of mycolic acids covalently attached to the cell wall and lower accumulation of free mycolic acids in the outer layer than the 410 strain. The low expression of latency proteins together with the reduced content of surface mycolic acids may facilitate Mp to evade the host immune responses.
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http://dx.doi.org/10.1155/2020/4741237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168692PMC
January 2021

Screening of inmates transferred to Spain reveals a Peruvian prison as a reservoir of persistent Mycobacterium tuberculosis MDR strains and mixed infections.

Sci Rep 2020 02 17;10(1):2704. Epub 2020 Feb 17.

Hospital General Universitario Gregorio Marañón. Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.

It is relevant to evaluate MDR-tuberculosis in prisons and its impact on the global epidemiology of this disease. However, systematic molecular epidemiology programs in prisons are lacking. A health-screening program performed on arrival for inmates transferred from Peruvian prisons to Spain led to the diagnosis of five MDR-TB cases from one of the biggest prisons in Latin America. They grouped into two MIRU-VNTR-clusters (Callao-1 and Callao-2), suggesting a reservoir of two prevalent MDR strains. A high-rate of overexposure was deduced because one of the five cases was coinfected by a pansusceptible strain. Callao-1 strain was also identified in 2018 in a community case in Spain who had been in the same Peruvian prison in 2002-5. A strain-specific-PCR tailored from WGS data was implemented in Peru, allowing the confirmation that these strains were currently responsible for the majority of the MDR cases in that prison, including a new mixed infection.
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http://dx.doi.org/10.1038/s41598-020-59373-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026066PMC
February 2020

Trends of Two Epidemic Multidrug-Resistant Strains of in Argentina Disclosed by Tailored Molecular Strategy.

Am J Trop Med Hyg 2019 12;101(6):1308-1311

Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.

Two strains-M (sublineage 4.1) and Ra (sublineage 4.3)-have long prevailed in Argentina among patients with multidrug-resistant tuberculosis (MDR-TB). Recently, budget constraints have hampered the surveillance of MDR-TB transmission. Based on whole-genome sequence analysis, we used M- and Ra-specific single nucleotide polymorphisms to tailor two multiplex allele-specific polymerase chain reactions (PCRs), which we applied to 252 stored isolates (95% of all newly diagnosed MDR-TB cases countrywide, 2015-2017). Compared with the latest data available (2007-2009), the M strain has receded (80/324 to 20/252, < 0.0001), particularly among cross-border migrants (12/58 to 0/53, = 0.0003) and HIV-infected people (30/97 to 7/74, = 0.0007), but it still accounts for 4/12 new cases of extensively drug-resistant TB. Differently, the Ra strain remained stable in frequency (39/324 to 33/252) and contributed marginally to the extensive drug-resistance load (1/12). Our novel strategy disclosed recent trends of the two major MDR-TB strains, providing meaningful data to allocate control interventions more efficiently.
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http://dx.doi.org/10.4269/ajtmh.19-0397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896870PMC
December 2019

Exploring the "Latin American Mediterranean" family and the RD lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela.

BMC Microbiol 2019 06 13;19(1):131. Epub 2019 Jun 13.

Laboratório de Biologia Molecular aplicada às Micobactérias, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, 21045-900, Brazil.

Background: The Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RD) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RD genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C SNP and the regions of difference RD and RD174. IS6110-RFLP results were also used when available.

Results: Genotyping of 413 M. tuberculosis isolates from three Latin-American countries detected LAM (46%) and the ill-defined T clade (16%) as the most frequent families. The highest clustering rate was detected in the sample population from the city of Caracas in Venezuela. We observed considerable differences in the presence of the RD lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C, MIRU02-MIRU40 signature) of the RD lineage were essentially confirmed. For the LAM family, the most polymorphic loci were MIRU40, MIRU31, MIRU10, MIRU26, MIRU16 and the least polymorphic MIRU24, MIRU20, MIRU04, MIRU23.

Conclusions: Our results suggest a differential adaptation of LAM-sublineages in neighboring populations and that RD strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RD) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.
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http://dx.doi.org/10.1186/s12866-019-1479-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567603PMC
June 2019

Survival of an epidemic MDR strain of Mycobacterium tuberculosis and its non-prosperous variant within activated macrophages.

Infect Genet Evol 2019 09 8;73:248-254. Epub 2019 May 8.

Instituto de Medicina Experimental (IMEX)-CONICET-Academia Nacional de Medicina, Pacheco de Melo 3081, C1425AUM Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, C1425FQB Buenos Aires, Argentina.

The fitness of a pathogen results from the interaction of multiple factors favoring either epidemiological success or failure. Herein, we studied the performance of the M strain, a highly successful multidrug resistant Mycobacterium tuberculosis genotype, and its non-prosperous variant, the 410 strain, in activated human monocyte-derived macrophages. Both strains showed comparable ability to induce necrotic cell death and to survive in apoptotic macrophages. Of the various macrophage activation conditions tested, none led to an enhanced control of the outbreak strain. The combination of 1,25(OH) vitaminD3 and IFN-γ favored significantly the control of the non-prosperous 410 strain. These observations indicate that the ability of the M strain to survive within the hostile intracellular milieu is conserved, and the overall fitness cost paid by this genotype would be low. Our results provide additional evidence on bacterial traits that may have contributed to the epidemiological success of the M strain.
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http://dx.doi.org/10.1016/j.meegid.2019.05.005DOI Listing
September 2019

Bedaquiline and linezolid MIC distributions and epidemiological cut-off values for Mycobacterium tuberculosis in the Latin American region.

J Antimicrob Chemother 2019 02;74(2):373-379

Université catholique de Louvain, Institute of Experimental and Clinical Research, Laboratory of Medical Microbiology, Brussels, Belgium.

Objectives: To describe the distributions of bedaquiline and linezolid MIC values for the Mycobacterium tuberculosis WT population and to define the corresponding epidemiological cut-offs (ECOFFs) in three Latin American countries.

Methods: MICs of bedaquiline and linezolid were determined by the resazurin microtitre assay (REMA). In phase 1, interlaboratory reproducibility was assessed using a panel of 10 fully susceptible M. tuberculosis strains. Phase 2 involved MIC determination for 248 clinical isolates from Argentina (n = 58), Brazil (n = 100) and Peru (n = 90) from patients who were treatment-naive for bedaquiline and linezolid. We then determined the ECOFFs for bedaquiline and linezolid by the eyeball method and the ECOFFinder statistical calculator.

Results: Phase 1: REMA MIC values in the three sites were either identical to each other or differed by one 2-fold dilution from the consensus value with the exception of a single value. Phase 2: the bedaquiline MIC range was 0.0039-0.25 mg/L for pan-susceptible and drug-resistant isolates combined. The linezolid MIC range was 0.062-0.5 mg/L for pan-susceptible isolates and 0.031-4 mg/L for drug-resistant isolates. ECOFFs were 0.125 mg/L for bedaquiline and 0.50 mg/L for linezolid.

Conclusions: REMA is reproducible and robust for the determination of bedaquiline and linezolid MIC distributions and ECOFF values when applied in laboratories of medium/low-resource countries. We suggest that WT MIC distributions for both drugs should be used as a monitoring tool to control the possible rapid emergence of resistance.
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http://dx.doi.org/10.1093/jac/dky414DOI Listing
February 2019

Global expansion of lineage 4 shaped by colonial migration and local adaptation.

Sci Adv 2018 10 17;4(10):eaat5869. Epub 2018 Oct 17.

Division of Infectious Diseases and Environmental Health, Norwegian Institute of Public Health, Lovisenberggata 8, 0456 Oslo, Norway.

On the basis of population genomic and phylogeographic analyses of 1669 lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.
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http://dx.doi.org/10.1126/sciadv.aat5869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192687PMC
October 2018

[One hundred years after the "Spanish" flu].

Medicina (B Aires) 2018 ;78(2):113-118

Comité de Redacción, Medicina (Buenos Aires), Buenos Aires, Argentina.

The "Spanish" flu pandemic, which occurred a century ago, is considered the most devastating in human history. An estimated one third of world population fell ill with flu and more than 2.5% of them died. The course of the epidemic had two main waves (1918 and 1919) and showed an unusual W-shaped morbidity/mortality distribution. Death was not a direct outcome of flu itself but rather a consequence of secondary bacterial bronchopneumonia, for which antibiotics had not yet been discovered. Pre-existing pulmonary tuberculosis was also accountable for increased flu death rates during the pandemic. As it happened in Europe, in Argentina the epidemic had two main waves, with ample variation in mortality by region. Available treatment at the time included diet, throat antiseptic rinses, low doses of quinine valerianate, salicylates, codeine as a cough suppressant, and camphor oil. Primitive anti-pneumococcal vaccines and immune sera were also applied. Upon the disclosure of the whole RNA sequence of the 1918 influenza virus genome, by means of reverse genetics it was possible to assemble viral particles resembling those of the deadly pandemic. The reconstituted virus proved to be extraordinarily virulent for mice. Current seasonal flu vaccines help to reduce, but not to abolish, the risk of another pandemic. The ongoing development of "universal" vaccines against influenza conferring reliable and long-lasting immunity may prevent its global spread in the future.
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February 2019

Genotypic diversity of Mycobacterium tuberculosis in Buenos Aires, Argentina.

Infect Genet Evol 2018 08 6;62:1-7. Epub 2018 Apr 6.

Instituto Nacional de Enfermedades Infecciosas INEI-ANLIS, Av. Vélez Sarsfield 563, Ciudad Autónoma de Buenos Aires C1282AFF, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas CONICET, Godoy Cruz 2290, Ciudad Autónoma de Buenos Aires C1425FQB, Argentina.

Buenos Aires is an overpopulated port city historically inhabited by people of European descent. Together with its broader metropolitan area, the city exhibits medium tuberculosis rates, and receives migrants, mainly from tuberculosis highly endemic areas of Argentina and neighboring countries. This work was aimed to gain insight into the Mycobacterium tuberculosis population structure in two suburban districts of Buenos Aires which are illustrative of the overall situation of tuberculosis in Argentina. The Lineage 4 Euro-American accounted for >99% of the 816 isolates analyzed (one per patient). Frequencies of spoligotype families were T 35.9%, LAM 33.2%, Haarlem 19.5%, S 3.2%, X 1.5%, Ural 0.7%, BOV 0.2%, Beijing 0.2%, and Cameroon 0.2%. Unknown signatures accounted for 5.3% isolates. Of 55 spoligotypes not matching any extant shared international type (SIT) in SITVIT database, 22 fitted into 15 newly-issued SITs. Certain autochthonous South American genotypes were found to be actively evolving. LAM3, which is wild type for RD, was the predominant LAM subfamily in both districts and the RD signature was rare among autochthonous, newly created, SITs and orphan patterns. Two genotypes that are rarely observed in neighboring countries ̶ SIT2/H2 and SIT159/T1 Tuscany ̶ were conspicuously represented in Argentina. The infrequent Beijing patterns belonged to Peruvian patients. We conclude that the genotype diversity observed reflects the influence of the Hispanic colonization and more recent immigration waves from Mediterranean and neighboring countries. Unlike in Brazil, the RD type does not play a major role in the tuberculosis epidemic in Buenos Aires.
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http://dx.doi.org/10.1016/j.meegid.2018.04.006DOI Listing
August 2018

Performance of a highly successful outbreak strain of Mycobacterium tuberculosis in a multifaceted approach to bacterial fitness assessment.

Int J Med Microbiol 2018 Apr 31;308(3):349-357. Epub 2018 Jan 31.

Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, C1425FQB, Buenos Aires, Argentina; Instituto Nacional de Enfermedades Infecciosas (INEI), ANLIS "Carlos G. Malbrán". Vélez Sarsfield 563, C1282AFF, Buenos Aires, Argentina. Electronic address:

Determining bacterial fitness represents a major challenge and no single parameter can accurately predict the ability of a certain pathogen to succeed. The M strain of Mycobacterium tuberculosis managed to spread and establish in the community and caused the largest multidrug-resistant tuberculosis outbreak in Latin America. We have previously shown that the M strain can manipulate the host immune response, but we still have no direct evidence, other than epidemiology, that can account for the enhanced fitness of the M strain. Our objective was to further characterize the performance of the outbreak strain M in different fitness assays. Two main aspects were evaluated: (1) molecular characterization of selected isolates from the M outbreak and related strains and (2) comparative fitness and in vivo performance of representative M strain isolates vs. the non-prosperous M strain variant 410. Our approach confirmed the multifaceted nature of fitness. Altogether, we conclude that the epidemiologically abortive strain 410 was vulnerable to drug-driven pressure, a weak competitor, and a stronger inductor of protective response in vivo. Conversely, the isolate 6548, representative of the M outbreak peak, had a growth disadvantage but performed very well in competition and induced lung damage at advanced stages in spite of reaching relatively low CFU counts. Integration of these observations supports the idea that the M strain managed to find a unique path to success.
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http://dx.doi.org/10.1016/j.ijmm.2018.01.006DOI Listing
April 2018

Multidrug-Resistant Strain M Induces Low IL-8 and Inhibits TNF- Secretion by Bronchial Epithelial Cells Altering Neutrophil Effector Functions.

Mediators Inflamm 2017 9;2017:2810606. Epub 2017 Aug 9.

Instituto de Medicina Experimental-CONICET-Academia Nacional de Medicina, Buenos Aires, Argentina.

M strain, the most prevalent multidrug-resistant strain of () in Argentina, has mounted mechanisms to evade innate immune response. The role of human bronchial epithelium in infection remains unknown as well as its crosstalk with neutrophils (PMN). In this work, we evaluate whether M and H37Rv strains invade and replicate within bronchial epithelial cell line Calu-6 and how conditioned media (CM) derived from infected cells alter PMN responses. We demonstrated that M infects and survives within Calu-6 without promoting death. CM from M-infected Calu-6 (M-CM) did not attract PMN in correlation with its low IL-8 content compared to H37Rv-CM. Also, PMN activation and ROS production in response to irradiated H37Rv were impaired after treatment with M-CM due to the lack of TNF-. Interestingly, M-CM increased H37Rv replication in PMN which would allow the spreading of mycobacteria upon PMN death and sustain IL-8 release. Thus, our results indicate that even at low invasion/replication rate within Calu-6, M induces the secretion of factors altering the crosstalk between these nonphagocytic cells and PMN, representing an evasion mechanism developed by M strain to persist in the host. These data provide new insights on the role of bronchial epithelium upon M infection.
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http://dx.doi.org/10.1155/2017/2810606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568625PMC
May 2018

Relation of Mycobacterium tuberculosis mutations at katG315 and inhA-15 with drug resistance profile, genetic background, and clustering in Argentina.

Diagn Microbiol Infect Dis 2017 Nov 28;89(3):197-201. Epub 2017 Jul 28.

Instituto Nacional de Enfermedades Infecciosas INEI-ANLIS, Buenos Aires, Argentina. Electronic address:

We analyzed 362 isoniazid-resistant clinical isolates of Mycobacterium tuberculosis obtained countrywide for the presence of mutation at katG315 and inhA-15 in relation to genotype, pattern of phenotypic resistance to other drugs, and ability to spread. We found the following mutation frequencies: katG315MUT/inhA-15wt 53.0%, katG315wt/inhA-15MUT 27.4%, katG315wt/inhA-15wt 19.3%, and katG315MUT/inhA-15MUT only 0.3%. Mutation at katG315 associated with the LAM superfamily; mutation at inhA-15 associated with the S family and the T1 Tuscany genotype; the combination katG315wt/inhA-15wt associated with the T1 Ghana genotype. Isolates harboring katG315MUT/inhA-15wt tended to accumulate resistance to other drugs and were more frequently found in cluster; isolates harboring katG315wt/inhA-15wt were more frequently found as orphan isolates. Although epidemiological and host factors could also be modulating the events observed, in Argentina, the systematic genotyping of drug resistant clinical isolates could help to predict an enhanced risk of transmission and a propensity to develop resistance to increasing numbers of drugs.
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http://dx.doi.org/10.1016/j.diagmicrobio.2017.07.010DOI Listing
November 2017

Single nucleotide polymorphisms may explain the contrasting phenotypes of two variants of a multidrug-resistant Mycobacterium tuberculosis strain.

Tuberculosis (Edinb) 2017 03 4;103:28-36. Epub 2017 Jan 4.

Universidad de Buenos Aires, Facultad de Agronomía, Cátedra de Microbiología Agrícola.INBA-CONICET, Av. San Martín 4453, C1417DSE, Buenos Aires, Argentina. Electronic address:

Globally, about 4.5% of new tuberculosis (TB) cases are multi-drug-resistant (MDR), i.e. resistant to the two most powerful first-line anti-TB drugs. Indeed, 480,000 people developed MDR-TB in 2015 and 190,000 people died because of MDR-TB. The MDR Mycobacterium tuberculosis M family, which belongs to the Haarlem lineage, is highly prosperous in Argentina and capable of building up further drug resistance without impairing its ability to spread. In this study, we sequenced the whole genomes of a highly prosperous M-family strain (Mp) and its contemporary variant, strain 410, which produced only one recorded tuberculosis case in the last two decades. Previous reports have demonstrated that Mp induced dysfunctional CD8 cytotoxic T cell activity, suggesting that this strain has the ability to evade the immune response against M. tuberculosis. Comparative analysis of Mp and 410 genomes revealed non-synonymous polymorphisms in eleven genes and five intergenic regions with polymorphisms between both strains. Some of these genes and promoter regions are involved in the metabolism of cell wall components, others in drug resistance and a SNP in Rv1861, a gene encoding a putative transglycosylase that produces a truncated protein in Mp. The mutation in Rv3787c, a putative S-adenosyl-l-methionine-dependent methyltransferase, is conserved in all of the other prosperous M strains here analysed and absent in non-prosperous M strains. Remarkably, three polymorphic promoter regions displayed differential transcriptional activity between Mp and 410. We speculate that the observed mutations/polymorphisms are associated with the reported higher capacity of Mp for modulating the host's immune response.
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http://dx.doi.org/10.1016/j.tube.2016.12.007DOI Listing
March 2017

C5aR contributes to the weak Th1 profile induced by an outbreak strain of Mycobacterium tuberculosis.

Tuberculosis (Edinb) 2017 03 21;103:16-23. Epub 2016 Dec 21.

Instituto de Medicina Experimental (IMEX) - CONICET, Academia Nacional de Medicina, Ciudad Autónoma de Buenos Aires, Argentina.

C5a anaphylatoxin is a component of the complement system involved in the modulation of T-cell polarization. Herein we investigated whether C5a receptors, C5aR and C5L2, modulate the cytokine profiles induced by Mycobacterium tuberculosis (Mtb). We analyzed the impact of both receptors on T helper cell polarization induced by the multidrug resistant outbreak strain named M, which is a poor IFN-γ inducer compared with the laboratory strain H37Rv. To this aim, we first blocked C5aR or C5L2 of peripheral blood monocytes (Mo) from patients with tuberculosis and healthy donors, then we stimulated the Mo either with H37Rv or the M strain, and finally we analyzed cytokine profiles of Mo/macrophages (MΦ) and CD4 T-cells. We found that: (i) Mtb modulated the expression of both C5a receptors, (ii) C5aR inhibited the expansion of CD4IFN-γ lymphocytes stimulated by the M strain but not by H37Rv, (iii) both receptors modulated the Mo/MΦ cytokine expression induced by Mtb. We conclude that C5aR, but not C5L2, plays a role in T helper cell polarization induced by Mtb and that this effect is strain- and donor-dependent. We speculate that the epidemiologically successful M strain takes advantage of this C5aR-mediated inhibition of Th1 polarization to survive within the host.
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http://dx.doi.org/10.1016/j.tube.2016.12.005DOI Listing
March 2017

Impact of HIV co-infection on the evolution and transmission of multidrug-resistant tuberculosis.

Elife 2016 08 9;5. Epub 2016 Aug 9.

UCL Genetics Institute, University College London, London, United Kingdom.

The tuberculosis (TB) epidemic is fueled by a parallel Human Immunodeficiency Virus (HIV) epidemic, but it remains unclear to what extent the HIV epidemic has been a driver for drug resistance in Mycobacterium tuberculosis (Mtb). Here we assess the impact of HIV co-infection on the emergence of resistance and transmission of Mtb in the largest outbreak of multidrug-resistant TB in South America to date. By combining Bayesian evolutionary analyses and the reconstruction of transmission networks utilizing a new model optimized for TB, we find that HIV co-infection does not significantly affect the transmissibility or the mutation rate of Mtb within patients and was not associated with increased emergence of resistance within patients. Our results indicate that the HIV epidemic serves as an amplifier of TB outbreaks by providing a reservoir of susceptible hosts, but that HIV co-infection is not a direct driver for the emergence and transmission of resistant strains.
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http://dx.doi.org/10.7554/eLife.16644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978521PMC
August 2016

Reactive oxygen species production by human dendritic cells involves TLR2 and dectin-1 and is essential for efficient immune response against Mycobacteria.

Cell Microbiol 2016 06 26;18(6):875-86. Epub 2016 Jan 26.

IMEX-CONTICET-ANM, Buenos Aires, Argentina and Servicio de Micobacterias, Instituto Malbrán, Buenos Aires, Argentina.

Tuberculosis remains the single largest infectious disease with 10 million new cases and two million deaths that are estimated to occur yearly, more than any time in history. The intracellular replication of Mycobacterium tuberculosis (Mtb) and its spread from the lungs to other sites occur before the development of adaptive immune responses. Dendritic cells (DC) are professional antigen-presenting cells whose maturation is critical for the onset of the protective immune response against tuberculosis disease and may vary depending on the nature of the cell wall of Mtb strain. Here, we describe the role of the endogenous production of reactive oxygen species (ROS) on DC maturation and expansion of Mtb-specific lymphocytes. Here, we show that Mtb induces DC maturation through TLR2/dectin-1 by generating of ROS and through Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) in a ROS independently manner. Based on the differences observed in the ability to induce DC maturation, ROS production and lymphocyte proliferation by those Mtb families widespread in South America, i.e., Haarlem and Latin American Mediterranean and the reference strain H37Rv, we propose that variance in ROS production might contribute to immune evasion affecting DC maturation and antigen presentation.
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http://dx.doi.org/10.1111/cmi.12562DOI Listing
June 2016

[Tuberculosis 110 years after the Nobel Prize awarded to Koch].

Medicina (B Aires) 2015 ;75(6):396-403

Instituto Nacional de Enfermedades Infecciosas-ANLIS Carlos Malbrán, Buenos Aires, Argentina.

The Nobel Prize in Physiology or Medicine was awarded in 1905 to Robert Koch "for his investigations and discoveries in relation to tuberculosis (TB)". He discovered the causal agent of TB, described the four principles that since then have guided research in communicable diseases and also prepared the old tuberculin, a bacillary extract that failed as a healing element but allowed the early diagnosis of TB infection and promoted the understanding of cellular immunity. After his death, the most conspicuous achievements against TB were the BCG vaccine, and the discovery of streptomycin, the antibiotic that launched the era of the effective treatment of TB. Drug-resistance soon appeared. In Argentina, studies on drug resistance began in the 60s. In the 70s, shortened anti-TB drug schemes were introduced consisting in two-month treatment with four drugs, followed by four months with two drugs. The incidence of TB decreased worldwide, but the immune depression associated with awarded together with the misuse of anti-TB drugs allowed the emergence of multidrug resistance and extensive resistance, with the emergence of nosocomial outbreaks worldwide, including Argentina. New rapid diagnostic methods based on molecular biology were developed and also new drugs, but the treatment of multidrug resistant and extensively resistant TB is still difficult and expensive. TB research has marked several milestones in medical sciences, including the monumental Koch postulates, the tuberculin skin test that laid the basis for understanding cell-mediated immunity, the first design of randomized clinical trials and the use of combined multi-drug treatments.
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June 2016

Four decades of transmission of a multidrug-resistant Mycobacterium tuberculosis outbreak strain.

Nat Commun 2015 May 11;6:7119. Epub 2015 May 11.

Department of Genetics, Evolution and Environment, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK.

The rise of drug-resistant strains is a major challenge to containing the tuberculosis (TB) pandemic. Yet, little is known about the extent of resistance in early years of chemotherapy and when transmission of resistant strains on a larger scale became a major public health issue. Here we reconstruct the timeline of the acquisition of antimicrobial resistance during a major ongoing outbreak of multidrug-resistant TB in Argentina. We estimate that the progenitor of the outbreak strain acquired resistance to isoniazid, streptomycin and rifampicin by around 1973, indicating continuous circulation of a multidrug-resistant TB strain for four decades. By around 1979 the strain had acquired additional resistance to three more drugs. Our results indicate that Mycobacterium tuberculosis (Mtb) with extensive resistance profiles circulated 15 years before the outbreak was detected, and about one decade before the earliest documented transmission of Mtb strains with such extensive resistance profiles globally.
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http://dx.doi.org/10.1038/ncomms8119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432642PMC
May 2015

Differential expression of immunogenic proteins on virulent Mycobacterium tuberculosis clinical isolates.

Biomed Res Int 2014 7;2014:741309. Epub 2014 Jul 7.

IMEX-CONICET, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 CABA, Argentina.

Molecular epidemiology has revealed that Mycobacterium tuberculosis (Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was also correlated with anti-Mtb-cell surface IgM, but not with anti-ManLAM, anti-PPD, or anti-Mtb-surface IgG antibodies. Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB.
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http://dx.doi.org/10.1155/2014/741309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109345PMC
April 2015

Outbreaks of Mycobacterium tuberculosis MDR strains differentially induce neutrophil respiratory burst involving lipid rafts, p38 MAPK and Syk.

BMC Infect Dis 2014 May 16;14:262. Epub 2014 May 16.

Inmunologia de enfermedades respiratorias, IMEX-CONTICET-ANM, Buenos Aires, Argentina.

Background: Neutrophils (PMN) are the first cells to infiltrate the lung after infection, and they play a significant protective role in the elimination of pathogen, by releasing preformed oxidants and proteolytic enzymes from granules and generating ROS, thus limiting inflammation by succumbing to apoptosis. In a previous study, we found marked differences in ROS-induced apoptosis between two Mycobacterium tuberculosis (Mtb) strains, M and Ra, representative of widespread Mtb families in South America, i.e. Haarlem and Latin-American Mediterranean (LAM), being strain M able to generate further drug resistance and to disseminate aggressively.

Methods: In this study we evaluate the nature of bacteria-PMN interaction by assessing ROS production, apoptosis, lipid raft coalescence, and phagocytosis induced by Mtb strains.

Results: Dectin-1 and TLR2 participate in Mtb-induced ROS generation and apoptosis in PMN involving p38 MAPK and Syk activation with the participation of a TLR2-dependent coalescence of lipid rafts. Further, ROS production occurs during the phagocytosis of non-opsonized bacteria and involves α-glucans on the capsule. In contrast, strain M lacks the ability to induce ROS because of: 1) a reduced phagocytosis and 2) a failure in coalescence of lipid raft.

Conclusions: The differences in wall composition could explain the success of some strains which stay unnoticed by the host through inhibition of apoptosis and ROS but making possible its replication inside PMN as a potential evasion mechanism. Innate immune responses elicited by Mtb strain-to-strain variations need to be considered in TB vaccine development.
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http://dx.doi.org/10.1186/1471-2334-14-262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049492PMC
May 2014

Mycobacterium tuberculosis multidrug resistant strain M induces an altered activation of cytotoxic CD8+ T cells.

PLoS One 2014 16;9(5):e97837. Epub 2014 May 16.

Laboratorio de Inmunología de Enfermedades Respiratorias, Instituto de Medicina Experimental-CONICET, Academia Nacional de Medicina, Buenos Aires, Argentina.

In human tuberculosis (TB), CD8+ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8+ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4+ and CD8+ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8+ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4+ and CD8+ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8+ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8+ T cells as well as CD4+ T cells collaboration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097837PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024032PMC
August 2015

Genotypes of Mycobacterium tuberculosis in patients at risk of drug resistance in Bolivia.

Infect Genet Evol 2013 Jul 17;17:195-201. Epub 2013 Apr 17.

Instituto Nacional de Enfermedades Infecciosas ANLIS Carlos G Malbrán, Vélez Sarsfield 563, 1281 Buenos Aires, Argentina.

Bolivia ranks among the 10 Latin American countries with the highest rates of tuberculosis (TB) and multidrug resistant (MDR) TB. In view of this, and of the lacking information on the population structure of Mycobacterium tuberculosis in the country, we explored genotype associations with drug resistance and clustering by analyzing isolates collected in 2010 from 100 consecutive TB patients at risk of drug resistance in seven of the nine departments in which Bolivia is divided. Fourteen isolates were MDR, 29 had other drug resistance profiles, and 57 were pansusceptible. Spoligotype family distribution was: Haarlem 39.4%, LAM 26.3%, T 22.2%, S 2.0%, X 1.0%, orphan 9.1%, with very low intra-family diversity and absence of Beijing genotypes. We found 66 different MIRU-VNTR patterns; the most frequent corresponded to Multiple Locus Variable Analysis (MLVA) MtbC15 patterns 860, 372 and 873. Twelve clusters, each with identical MIRU-VNTR and spoligotypes, gathered 35 patients. We found no association of genotype with drug resistant or MDR-TB. Clustering associated with SIT 50 and the H3 subfamily to which it belongs (p<0.0001). The largest cluster involved isolates from three departments and displayed a genotype (SIT 50/MLVA 860) previously identified in Bolivian migrants into Spain and Argentina suggesting that this genotype is widespread among Bolivian patients. Our study presents a first overview of M. tuberculosis genotypes at risk of drug resistance circulating in Bolivia. However, results should be taken cautiously because the sample is small and includes a particular subset of M. tuberculosis population.
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http://dx.doi.org/10.1016/j.meegid.2013.04.010DOI Listing
July 2013

Two genetically-related multidrug-resistant Mycobacterium tuberculosis strains induce divergent outcomes of infection in two human macrophage models.

Infect Genet Evol 2013 Jun 24;16:151-6. Epub 2013 Jan 24.

Instituto de Medicina Experimental (IMEX) - CONICET, Academia Nacional de Medicina, Pacheco de Melo 3081, (C1425ASU) Buenos Aires, Argentina.

Mycobacterium tuberculosis has a considerable degree of genetic variability resulting in different epidemiology and disease outcomes. We evaluated the pathogen-host cell interaction of two genetically closely-related multidrug-resistant M. tuberculosis strains of the Haarlem family, namely the strain M, responsible for an extensive multidrug-resistant tuberculosis outbreak, and its kin strain 410 which caused a single case in two decades. Intracellular growth and cytokine responses were evaluated in human monocyte-derived macrophages and dU937 macrophage-like cells. In monocyte-derived macrophages, strain M grew more slowly and induced lower levels of TNF-α and IL-10 than 410, contrasting with previous studies with other strains, where a direct correlation was observed between increased intracellular growth and epidemiological success. On the other hand, in dU937 cells, no difference in growth was observed between both strains, and strain M induced significantly higher TNF-α levels than strain 410. We found that both cell models differed critically in the expression of receptors for M. tuberculosis entry, which might explain the different infection outcomes. Our results in monocyte-derived macrophages suggest that strain M relies on a modest replication rate and cytokine induction, keeping a state of quiescence and remaining rather unnoticed by the host. Collectively, our results underscore the impact of M. tuberculosis intra-species variations on the outcome of host cell infection and show that results can differ depending on the in vitro infection model.
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http://dx.doi.org/10.1016/j.meegid.2013.01.007DOI Listing
June 2013

HIV infection and geographically bound transmission of drug-resistant tuberculosis, Argentina.

Emerg Infect Dis 2012 Nov;18(11):1802-10

Instituto Nacional de Enfermedades Infecciosas ANLIS Dr. Carlos G. Malbrán Buenos Aires, Argentina.

During 2003-2009, the National Tuberculosis (TB) Laboratory Network in Argentina gave 830 patients a new diagnosis of multidrug-resistant (MDR) TB and 53 a diagnosis of extensively drug- resistant (XDR) TB. HIV co-infection was involved in nearly one third of these cases. Strain genotyping showed that 7 major clusters gathered 56% of patients within restricted geographic areas. The 3 largest clusters corresponded to epidemic MDR TB strains that have been undergoing transmission for >10 years. The indigenous M strain accounted for 29% and 40% of MDR and XDR TB cases, respectively. Drug-resistant TB trends in Argentina are driven by spread of a few strains in hotspots where the rate of HIV infection is high. To curb transmission, the national TB program is focusing stringent interventions in these areas by strengthening infection control in large hospitals and prisons, expediting drug resistance detection, and streamlining information-sharing systems between HIV and TB programs.
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http://dx.doi.org/10.3201/eid1811.120126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559153PMC
November 2012