Strain Classification of Mycobacterium tuberculosis Isolates in Brazil Based on Genotypes Obtained by Spoligotyping, Mycobacterial Interspersed Repetitive Unit Typing and the Presence of Large Sequence and Single Nucleotide Polymorphism

International audienceRio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as "LAM-like", 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

The changing face of the epidemiology of tuberculosis due to molecular strain typing - A Review

About one third of the world population is infected with tubercle bacilli, causing eight million new cases of tuberculosis (TB) and three million deaths each year. After years of lack of interest in the disease, World Health Organization recently declared TB a global emergency and it is clear that there is need for more efficient national TB programs and newly defined research priorities. A more complete epidemiology of tuberculosis will lead to a better identification of index cases and to a more efficient treatment of the disease. Recently, new molecular tools became available for the identification of strains of Mycobacterium tuberculosis (M. tuberculosis), allowing a better recognition of transmission routes of defined strains. Both a standardized restriction-fragment-length-polymorphism-based methodology for epidemiological studies on a large scale and deoxyribonucleic acids (DNA) amplification-based methods that allow rapid detection of outbreaks with multidrug-resistant (MDR) strains, often characterized by high mortality rates, have been developed. This review comments on the existing methods of DNA-based recognition of M. tuberculosis strains and their peculiarities. It also summarizes literature data on the application of molecular fingerprinting for detection of outbreaks of M. tuberculosis, for identification of index cases, for study of interaction between TB and infection with the human immunodeficiency virus, for analysis of the behavior of MDR strains, for a better understanding of risk factors for transmission of TB within communities and for population-based studies of TB transmission within and between countries

The changing face of the epidemiology of tuberculosis due to molecular strain typing--a review

Submitted by Sandra Infurna ([email protected]) on 2019-11-02T19:55:44Z No. of bitstreams: 1 PhilipSuffys_WinDegrave_etal_IOC_1997.pdf: 1105196 bytes, checksum: de8f7ff823c6fd869e41c3f0ed356c1f (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-11-02T20:05:03Z (GMT) No. of bitstreams: 1 PhilipSuffys_WinDegrave_etal_IOC_1997.pdf: 1105196 bytes, checksum: de8f7ff823c6fd869e41c3f0ed356c1f (MD5)Made available in DSpace on 2019-11-02T20:05:03Z (GMT). No. of bitstreams: 1 PhilipSuffys_WinDegrave_etal_IOC_1997.pdf: 1105196 bytes, checksum: de8f7ff823c6fd869e41c3f0ed356c1f (MD5) Previous issue date: 1997Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Laboratório de Biologia Molecular e Diagnóstico de Doenças Infecciosas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Laboratório de Biologia Molecular e Diagnóstico de Doenças Infecciosas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Laboratório de Biologia Molecular e Diagnóstico de Doenças Infecciosas. Rio de Janeiro, RJ, Brasil.About one third of the world population is infected with tubercle bacilli, causing eight million new cases of tuberculosis (TB) and three million deaths each year. After years of lack of interest in the disease, World Health Organization recently declared TB a global emergency and it is clear that there is need for more efficient national TB programs and newly defined research priorities. A more complete epidemiology of tuberculosis will lead to a better identification of index cases and to a more efficient treatment of the disease. Recently, new molecular tools became available for the identification of strains of Mycobacterium tuberculosis (M. tuberculosis), allowing a better recognition of transmission routes of defined strains. Both a standardized restriction-fragment-length-polymorphism-based methodology for epidemiological studies on a large scale and deoxyribonucleic acids (DNA) amplification-based methods that allow rapid detection of outbreaks with multidrug-resistant (MDR) strains, often characterized by high mortality rates, have been developed. This review comments on the existing methods of DNA-based recognition of M. tuberculosis strains and their peculiarities. It also summarizes literature data on the application of molecular fingerprinting for detection of outbreaks of M. tuberculosis, for identification of index cases, for study of interaction between TB and infection with the human immuno-deficiency virus, for analysis of the behavior of MDR strains, for a better understanding of risk factors for transmission of TB within communities and for population-based studies of TB transmission within and between countries

Frequencies of strains according to classification by Spoligotyping (SITVIT2) and 24 loci MIRU-VNTR (MIRUVNTRPlus).

<p>Frequencies of strains according to classification by Spoligotyping (SITVIT2) and 24 loci MIRU-VNTR (MIRUVNTRPlus).</p

The frequency of <i>fbpC</i>¹⁰³, RD^Rio and RD174 in LAM, LAM-like and Non LAM isolates, as designated by spoligotyping and 24 MIRU-VNTR typing.

<p>The frequency of <i>fbpC</i>¹⁰³, RD^Rio and RD174 in LAM, LAM-like and Non LAM isolates, as designated by spoligotyping and 24 MIRU-VNTR typing.</p

Description of clusters containing 3 or more isolates in this study and their worldwide distribution in the SITVIT2 database (interrogation made on September 25^th 2013).

<p>*Worldwide distribution is reported for regions with more than 3% of a given SITs as compared to their total number in the SITVIT2 database. The definition of macro-geographical regions and sub-regions (<a href="http://unstats.un.org/unsd/methods/m49/m49regin.htm" target="_blank">http://unstats.un.org/unsd/methods/m49/m49regin.htm</a>) is according to the United Nations; Regions: AFRI (Africa), AMER (Americas), ASIA (Asia), EURO (Europe), and OCE (Oceania), subdivided in: E (Eastern), M (Middle), C (Central), N (Northern), S (Southern), SE (South-Eastern), and W (Western). Furthermore, CARIB (Caribbean) belongs to Americas, while Oceania is subdivided in 4 sub-regions, AUST (Australasia), MEL (Melanesia), MIC (Micronesia), and POLY (Polynesia). Note that in our classification scheme, Russia has been attributed a new sub-region by itself (Northern Asia) instead of including it among rest of the Eastern Europe. It reflects its geographical localization as well as due to the similarity of specific TB genotypes circulating in Russia (a majority of Beijing genotypes) with those prevalent in Central, Eastern and South-Eastern Asia.</p><p>**The 3 letter country codes are according to <a href="http://en.wikipedia.org/wiki/ISO_3166-1_alpha-3" target="_blank">http://en.wikipedia.org/wiki/ISO_3166-1_alpha-3</a>; countrywide distribution is only shown for SITs with ≥3% of a given SITs as compared to their total number in the SITVIT2 database.</p><p>Description of clusters containing 3 or more isolates in this study and their worldwide distribution in the SITVIT2 database (interrogation made on September 25^th 2013).</p

Venn diagram illustrating the different markers in isolated classified as LAM by 24 loci MIRU-VNTR.

<p>Notes: The Venn diagram was constructed based on LAM isolates defined by 24 loci MIURU-VNTR. The sizes of the circles is not proportional to the real frequency of these markers. Twenty isolates classified with LAM by MIRU-VNTRs were removed from the final analysis because of showing a mixed genotype or failure in at least one of the three genotype procedures. * absence of spacers 33–36 in the spoligotyping profile, ** absence of spacers 21–24 in the spoligotyping profile, ***Two isolates RD^Rio but not RD174 (LAM2 SIT3908).</p

Confusion Matrix comparing the classifications obtained by Spoligotyping and MIRU-VNTR. ¹

<p>Classification according to SITIVIT2. For unknown Spoligotypes, we used SpotClust. ² Patterns were classified based on a VNTR<i>plus</i> database that allows construction of a Neighbor-Joining based phylogenetic tree, visualizing proximity of a particular genotype with that of a set of reference strains to the genotype family level.</p