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

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.

Association between bacterial homoplastic variants and radiological pathology in tuberculosis.

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 Mycobacterium tuberculosis genome. METHODS: We performed whole genome sequencing (Illumina HiSeq2000 platform) on M. tuberculosis 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 M. tuberculosis 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.

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

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.

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

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 (RDRio) 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 RDRio 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 Ag85C103 SNP and the regions of difference RDRio 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 RDRio lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C103, MIRU02-MIRU40 signature) of the RDRio 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 RDRio strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RDRio) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.

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

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.

[One hundred years after the "Spanish" flu]. / A cien años de la gripe "española".

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.

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

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.

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

M strain, the most prevalent multidrug-resistant strain of Mycobacterium tuberculosis (Mtb) in Argentina, has mounted mechanisms to evade innate immune response. The role of human bronchial epithelium in Mtb 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-α. 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.

[Tuberculosis 110 years after the Nobel Prize awarded to Koch]. / La tuberculosis a 110 años del Premio Nobel de Koch.

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.

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

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.

IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes.

BACKGROUND: The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment. RESULTS: We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed. CONCLUSIONS: This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.

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

Successful alternative treatment of extensively drug-resistant tuberculosis in Argentina with a combination of linezolid, moxifloxacin and thioridazine.

OBJECTIVES: Current drug choices to treat extensively drug-resistant (XDR) tuberculosis (TB) are scarce; therefore, information on the safety, tolerability and efficacy of alternative regimens is of utmost importance. The aim of this study was to describe the management, drug adverse effects and outcome of alternative combined treatment in a series of XDR-TB patients. PATIENTS AND METHODS: A retrospective study was performed on 17 non-AIDS, pulmonary adult patients with XDR-TB admitted to a referral treatment centre for infectious diseases in Buenos Aires from 2002 through 2008. Drug susceptibility testing was performed under regular proficiency testing and confirmed at the national TB reference laboratory. RESULTS: Linezolid was included in the drug regimens of all patients; moxifloxacin and/or thioridazine were included in the regimens of 14 patients. Clinically tractable drug adverse effects were observed in nine patients, the most frequent being haematological disorders and neurotoxicity. In two patients, thioridazine was discontinued. Negative culture conversion was achieved in 15 patients, 11 completed treatment meeting cure criteria, 4 are still on follow-up with good evolution, 1 defaulted treatment and 1 was lost to follow-up. CONCLUSIONS: The combination of linezolid, moxifloxacin and thioridazine is recommended for compassionate use in specialized centres with expertise in the management of XDR-TB.

Clinical isolates of Mycobacterium tuberculosis differ in their ability to induce respiratory burst and apoptosis in neutrophils as a possible mechanism of immune escape.

Tuberculosis pathogenesis was earlier thought to be mainly related to the host but now it appears to be clear that bacterial factors are also involved. Genetic variability of Mycobacterium tuberculosis (Mtb) could be slight but it may lead to sharp phenotypic differences. We have previously reported that nonopsonized Mtb H37Rv induce apoptosis of polymorphonuclear neutrophils (PMNs) by a mechanism that involves the p38 pathway. Here we evaluated the capability to induce PMN apoptosis of two prevalent Mtb lineages in Argentina, the Latin America and Mediterranean (LAM), and Haarlem, using the H37Rv as a reference strain. Results showed that LAM strains strongly induced apoptosis of PMN which correlated with the induction of reactive oxygen species (ROS) production and p38 activation. Interestingly, the highly prosperous multidrug-resistant M strain, belonging to the Haarlem lineage, lacked the ability to activate and to induce PMN apoptosis as a consequence of (1) a weak ROS production and (2) the contribution of antiapoptotic mechanisms mediated at least by ERK. Although with less skill, M is able to enter the PMN so that phenotypic differences could lead PMN to be a reservoir allowing some pathogens to prevail and persist over other strains in the community.

Outbreaks of mycobacterium tuberculosis MDR strains induce high IL-17 T-cell response in patients with MDR tuberculosis that is closely associated with high antigen load.

BACKGROUND: The proinflammatory cytokine interleukin 17 (IL-17) plays an important role in immune responses but it is also associated with tissue-damaging inflammation. So, we evaluated the ability of Mycobacterium tuberculosis clinical isolates to induce IL-17 in tuberculosis (TB) patients and in healthy human tuberculin reactors (PPD(+)HD). METHODS: IL-17, interferon γ (IFN-γ), and interleukin 23 (IL-23) receptor expression were evaluated ex vivo and cultured peripheral blood mononuclear cells from TB and PPD(+)HD stimulated with irradiated clinical isolates from multidrug resistant (MDR) outbreaks M (Haarlem family) and Ra (Latin American-Mediterranean family), as well as drug-susceptible isolates belonging to the same families and laboratory strain H37Rv for 48 hours in T-cell subsets by flow cytometry. RESULTS: We observed that: (1) MDR strains M and Ra are stronger IL-17 inducers than drug-susceptible Mtb strains of the Haarlem and Latin American-Mediterranean families, (2) MDR-TB patients show the highest IL-17 expression that is independent on the strain, (3) IL-17 expression is dependent on CD4(+) and CD8(+) T cells associates with persistently high antigen load. CONCLUSIONS: IL-17--producing T cells could play an immunopathological role in MDR-TB promoting severe tissue damage, which may be associated with the low effectiveness of the second-line drugs employed in the treatment.

The host-pathogen-environment triad: Lessons learned through the study of the multidrug-resistant Mycobacterium tuberculosis M strain.

Multidrug-resistant tuberculosis is one of the major obstacles that face the tuberculosis eradication efforts. Drug-resistant Mycobacterium tuberculosis clones were initially disregarded as a public health threat, because they were assumed to have paid a high fitness cost in exchange of resistance acquisition. However, some genotypes manage to overcome the impact of drug-resistance conferring mutations, retain transmissibility and cause large outbreaks. In Argentina, the HIV-AIDS epidemics fuelled the expansion of the so-called M strain in the early 1990s, which is responsible for the largest recorded multidrug-resistant tuberculosis cluster of Latin America. The aim of this work is to review the knowledge gathered after nearly three decades of multidisciplinary research on epidemiological, microbiological and immunological aspects of this highly successful strain. Collectively, our results indicate that the successful transmission of the M strain could be ascribed to its unaltered virulence, low Th1/Th17 response, a low fitness cost imposed by the resistance conferring mutations and a high resistance to host-related stress. In the early 2000s, the incident cases due to the M strain steadily declined and stabilized in the latest years. Improvements in the management, diagnosis and treatment of multidrug-resistant tuberculosis along with societal factors such as the low domestic and international mobility of the patients affected by this strain probably contributed to the outbreak containment. This stresses the importance of sustaining the public health interventions to avoid the resurgence of this conspicuous multidrug-resistant strain.

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

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.

The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: providing guidelines for Quality Assurance when working on membranes.

BACKGROUND: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. METHODS: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. RESULTS: Seven out of the 11 laboratories (63%), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). CONCLUSIONS: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping.

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

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.

Los virus ARN, excepto los retrovirus, se replican por acción de una ARN polimerasa ARN-dependiente que carece de exonucleasa correctora y, en consecuencia, en cada replicación puede cometer errores. Así se originan mutantes que, según su menor o mayor fitness, se extinguen o bien prosperan y originan variantes que escapan al sistema inmune. Las mutaciones de SARS-CoV-2 más importantes son las que alteran la proteína viral S, porque ella tiene la llave de ingreso del virus a la célula humana. Cuanto más se replican los virus, más mutan, y se hace más probable que aparezcan variantes resistentes dominantes. En esos casos, se requerirá una aplicación más estricta de las medidas de protección de la comunidad. Las vacunas y los anticuerpos policlonales, que inducen una respuesta dirigida hacia toda la proteína S, mantendrían protección efectiva contra las variantes del SARSCoV-2. Además, las vacunas inducirían una mayor respuesta de células T helper y citotóxicas, lo que puede ser un biomarcador de protección. En áreas densamente pobladas con escasas medidas de protección, el virus se difunde libremente y aumenta la probabilidad de mutaciones de escape. India y Manaos ejemplifican esa situación. La evolución natural selecciona las mutantes que se reproducen con mayor eficiencia sin eliminar al huésped, lo que facilita la propagación. En cambio, la circulación de virus de alta virulencia y letalidad (Ebola, hantavirus), que eliminan al huésped, se circunscribe a determinadas áreas geográficas, sin mayor difusión. Por lo tanto, sería esperable que SARS-CoV-2 evolucione a variantes más infecciosas y menos virulentas.

One hundred years of BCG vaccine.

The BCG vaccine was given for the first time in 1921, in Paris, to a newborn of a mother with tuberculosis. Between 1924 and 1960, the Pasteur Institute delivered BCG cultures to more than 50 laboratories around the world. In 1925, Dr Andrés Arena introduced the BCG seed to Argentina, where the vaccine began to be produced and applied orally to newborns. The original strain underwent diverse genetic changes in different parts of the world, which did not seem to affect its protective efficacy. In Argentina, a study (1978-1985) showed that BCG prevents primary TB in general, and has 100% efficacy in meningitis and other extra-pulmonary TB locations. BCG effect is independent of TB control measures (case detection and treatment). Furthermore, BCG provides nonspecific protection from various infections and is used in the treatment of bladder cancer. By 2020, at least five technologies had already been established for the future development of anti-TB vaccines: cellular vaccines, protein subunits, nucleic acids, with adenovirus vector, and with recombinant influenza virus as a vector. There are currently more than 20 TB vaccine candidates under evaluation. History teaches, and the COVID-19 pandemic has confirmed, that vaccination is a fundamental instrument for the control of infectious diseases. Until a more effective vaccine becomes available, BCG will continue to be included in the Argentine National Vaccination Calendar for application to newborns.

La vacuna BCG fue administrada por primera vez en 1921, en París, a un recién nacido de madre tuberculosa. Entre 1924 y 1960, el Instituto Pasteur entregó cultivos de BCG a más de 50 laboratorios de todo el mundo. En 1925, el Dr. Andrés Arena lo introdujo en Argentina, donde se comenzó a producir y aplicar la vacuna a recién nacidos por vía oral. La cepa original sufrió múltiples cambios genéticos que, sin embargo, no parecen haber afectado su eficacia protectora, establecida aun sin que se conociera el mecanismo de acción. En Argentina, un estudio (1978-1985) demostró que la BCG previene la TB primaria en general, y en un 100% la meningitis y otras localizaciones extrapulmonares. Su efecto es independiente de las medidas de control de la TB (detección de casos y tratamiento). Además, se la usa en el tratamiento del cáncer de vejiga y provee protección inespecífica contra diversas enfermedades infecciosas. En 2020 ya se habían establecido por lo menos 5 tecnologías para el futuro desarrollo de vacunas anti-TB: vacunas celulares, de subunidades proteicas, de ácidos nucleicos, con vector adenovirus, y con virus influenza recombinante como vector. Actualmente hay más de 20 vacunas candidatas anti-TB. La historia enseña, y la pandemia de COVID-19 ha contribuido a revalorizar, que la vacunación es un instrumento fundamental para el control y la erradicación de las enfermedades infecciosas. Y hasta que haya disponible otra más eficaz, BCG seguirá figurando en el Calendario de Vacunación Nacional, para ser aplicada al recién nacido.