Papers by Maranibia Oelemann
<p>A selection of 137 isolates was used, representing the diversity of the different lineag... more <p>A selection of 137 isolates was used, representing the diversity of the different lineages and subgroups predicted based on MIRU-VNTR typing, spoligotyping and the MIRU-VNTR<i>Plus</i> database (see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-g001" target="_blank">Fig. 1</a>). A. A MIRU-VNTR-based dendrogram was generated using the neighbor-joining algorithm and rooted using a <i>M. prototuberculosis</i> C/D genotype (alias <i>M. canettii</i>) as outgroup. Solid coloured circles on tree nodes indicate MIRU-VNTR groupings that are monophyletic when compared to LSP-based (deligotyping) and/or (when deligotyping was not informative) spoligotyping-based groupings. Partially monophyletic groupings are indicated by coloured rings. B. MIRU-VNTR-based minimum spanning tree. The same isolates were used as in the neighbor-joining tree. Colours and grouping names correspond to those of panel A. Distances between circles are proportional to the number of allele differences between MIRU-VNTR genotypes; circle sizes are proportional to the numbers of isolates sharing an identical genotype. Del, deligotyping probe; RD, region of difference (reference LSP, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-t001" target="_blank">Table 1</a>); spol, spoligotyping spacer; LAMu, unclassified LAM isolate. Color codes of phylogenetic groups (see text for further description): yellow, Indo-Oceanic (LSP)/EAI (spoligotyping); grey, S; light purple, X; red, Haarlem; blue, Brazil 1; dark purple; Brazil 2; pink, LAM II; khaki, LAM I; green, LAM III.</p
<p>Discriminatory powers of different genotyping methods used.</p
<p>Molecular clusters are defined by isolates sharing identical 24-locus MIRU-VNTR genotype... more <p>Molecular clusters are defined by isolates sharing identical 24-locus MIRU-VNTR genotypes and spoligotypes.</p
<p>Colour-coded 24-locus MIRU-VNTR alleles and spoligotypes from 361 isolates are represent... more <p>Colour-coded 24-locus MIRU-VNTR alleles and spoligotypes from 361 isolates are represented. A MIRU-VNTR-based dendrogram was generated using the neighbor-joining algorithm and rooted using a <i>M. prototuberculosis</i> C/D genotype (alias <i>M. canettii</i>) as outgroup. <i>M. tuberculosis</i> strain lineages and branches shown at the right were identified by analyzing the congruence of MIRU-VNTR typing and spoligotyping results within this collection and submitting the isolate genotypes to the MIRU-VNTR<i>Plus</i> identification database (see text). X gr1 and gr2 (groups 1 and 2) correspond to X del26/RD183 and Xdel29/RD193 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-g003" target="_blank">Fig. 3</a>, respectively.</p
Inst Oswaldo Cruz, Dept Mycobacteriosis, Lab Mol Biol Appl Mycobacteria, BR-20001 Rio de Janeiro,... more Inst Oswaldo Cruz, Dept Mycobacteriosis, Lab Mol Biol Appl Mycobacteria, BR-20001 Rio de Janeiro, Brazil
PLoS ONE, 2011
Background: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiologi... more Background: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. Methodology/Principal Findings: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. Conclusions/Significance: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications.
Background: Gene deletion in M. tuberculosis (Mtb) is emerging as determinant of biological diver... more Background: Gene deletion in M. tuberculosis (Mtb) is emerging as determinant of biological diversity. Identification of deletion might help to understand the role of specific genes and serve as markers for epidemiological studies. Methods: As an extension of previous work (JCM 2003; 41:1637-50), 406 clinical isolates from 309 patients with tuberculosis collected in 2002 and 2003 from Rio de Janeiro (RJ), Brazil, were tested using the our PCR panel and molecularly typed by principal genetic group (PGG), spoligotyping and MIRU. Results: 30% of the Mtb isolates unexpectedly failed to amplify the IS1561’ locus. PCR successfully bridge the deleted segment for all the IS1561’ deleted Mtb strains (n=124) but none of the IS1561’-positive Mtb strains (n=282); they were named RDRio and Wildtype (WT) strains, respectively. Sequence analysis suggested that homologous recombination was the mechanism responsible for the deletion of unprecedented length of >26 Kb. Among the genes deleted there...
Memórias do Instituto Oswaldo Cruz, 2013
The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB).... more The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.
Journal of Microbiological Methods, 2003
The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency ... more The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n = 55), H526Y (n = 9), H526D (n = 12) or D516V (n = 9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution. D
Infection, Genetics and Evolution, 2012
One of the high tuberculosis (TB) incidence countries in the world, Brazil is characterized by co... more One of the high tuberculosis (TB) incidence countries in the world, Brazil is characterized by considerable differences in TB incidence on regional and state level. In the present study, we describe Brazilian spoligotypes of 1991 Mycobacterium tuberculosis complex (MTC) clinical isolates from patients residents of 11 states from different regions of the country, diagnosed between 1996 and 2005. By performing spoligotyping on a large number of M. tuberculosis clinical isolates, one of the main objectives of this study was to determine the major genotype families causing TB in Brazil and to verify the region-associated genotype distribution. We observed a total of 577 distinct spoligopatterns, 12.6% of these corresponded to orphan patterns while 87.4% belonged to 326 shared-types (SITs). Among the latter, 86 SITs (isolated from 178 patients) had been observed for the first time in this study, the most frequent being SIT2517 which belonged to the T3-ETH lineage and was exclusively found among patients residents of Belém, the capital of the state of Pará (n = 8 isolates). Irrespective of shared-type labeling, a total of 19.5% strains were unique (unclustered) in our study as opposed to 80.5% clustered isolates (189 clusters, size range from 2 to 205 isolates). The three largest clusters were SIT42 of the Latin-America & Mediterranean (LAM) 9 clade (10.3%), SIT53 of the T clade (7.6%), and SIT50 of the Haarlem clade (5.4%). The predominant MTC lineages in Brazil in decreasing order belonged to the LAM (46%); the ill-defined T (18.6%); the Haarlem (12.2%), the X (4.7%), the S (1.9%), and the East African Indian (EAI) (0.85%) families. The rest of clades grouped together as Mycobacterium africanum, Mycobacterium bovis, Beijing, Central Asian (CAS), and the Manu types, represented less than 1% of the strains. Finally, about 15% of the isolates showed spoligotype signatures that were not yet classified among well-defined lineages. In conclusion, we provide hereby a first insight into the population structure of MTC isolates in Brazil, showing the predominance of both LAM and T family and the existence of region-associated genotypes.
Memórias do Instituto Oswaldo Cruz
Memórias do Instituto Oswaldo Cruz, 2007
Fingerprinting of Mycobacterium tuberculosis strains from tuberculosis (TB) patients attended in ... more Fingerprinting of Mycobacterium tuberculosis strains from tuberculosis (TB) patients attended in Community Health Centers (CHCs) of Rio de Janeiro was performed to verify possible risk factors for TB transmission. A prospective community-based study was performed during the period of July 1996 to December 1996 by collecting sputum samples of 489 patients in 11 different CHCs in four different planning areas (APs) of the city. Bacteriological, clinical, and epidemiological information was collected and M. tuberculosis genotypes defined after restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element (DRE) fingerprinting of RFLP-clustered cases. Risk factors for TB transmission were looked for using three levels of cluster stringency. Among 349 (71%) positive cultures obtained, IS6110-RFLP typing could be performed on strains from 153 different patients. When using identity of RFLP patterns as cluster definition, 49 (32%) of the strains belonged to a cluster and none of the clinical or epidemiologic characteristics was associated with higher clustering levels. However, higher clustering level was observed in the AP including the central region of the city when compared to others. This strongly suggests that more recent transmission occurs in that area and this may be related with higher incidence of TB and HIV in this region.
Memórias do Instituto Oswaldo Cruz, 2012
We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tand... more We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil. Romano MI 1999. Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis -related infections in South America. J Clin Microbiol 37: 296-303.
Memórias do Instituto Oswaldo Cruz, 2002
Little is known about transmission and drug resistance of tuberculosis (TB) in Bauru, State of S ... more Little is known about transmission and drug resistance of tuberculosis (TB) in Bauru, State of S o Paulo. The objective of this study was to evaluate risk factors for transmission of Mycobacterium tuberculosis strains in this area. Strains were collected from patients attended at ambulatory services in the region and susceptibility towards the main first line antibiotics was determined and fingerprinting performed. A total of 57 strains were submitted to susceptibility testing: 23 (42.6%) were resistant to at least one drug while 3 (13%) were resistant against both rifampicin and isoniazide. Resistant strains had been isolated from patients that had not (n = 13) or had (n = 9) previously been submitted to anti-TB treatment, demonstrating a preoccupying high level of primary resistance in the context of the study. All strains were submitted to IS6110 restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element PCR (DRE-PCR). Using IS6110-RFLP, 26.3% of the strains were clustered and one cluster of 3 patients included 2 HIV-infected individuals that had been hospitalized together during 16 days; clustering of strains of patients from the hospital was however not higher than that of patients attended at health posts. According to DRE-PCR, 55.3% belonged to a cluster, confirming the larger discriminatory power of IS6110-RFLP when compared to DRE-PCR, that should therefore be used as a screening procedure only. No clinical, epidemiological or microbiological characteristics were associated with clustering so risk factors for transmission of TB could not be defined in the present study.
Journal of Clinical Microbiology, 2007
The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for ... more The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for genomic deletions. One locus in our panel of PCR targets failed to amplify in ϳ30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RD Rio . Homologous recombination between two similar protein-coding genes is proposed as the mechanism for deleting or modifying 10 genes, including two potentially immunogenic PPE proteins. The flanking regions of the RD Rio locus were identical in all strains bearing the deletion. Genetic testing by principal genetic group, spoligotyping, variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR), and IS6110-based restriction fragment length polymorphism analysis cumulatively support the idea that RD Rio strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RD Rio lineage is therefore the predominant clade causing tuberculosis (TB) in Rio de Janeiro and, as indicated by genotypic clustering in MIRU-VNTR analysis, the most significant source of recent transmission. Limited retrospective reviews of bacteriological and patient records showed a lack of association with multidrug resistance or specific risk factors for TB. However, trends in the data did suggest that RD Rio strains may cause a form of TB with a distinct clinical presentation. Overall, the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a Euro-Latin American host population. The identification of RD Rio strains outside of Brazil points to the ongoing intercontinental dissemination of this important genotype. Further studies are needed to determine the differential strain-specific features, pathobiology, and worldwide prevalence of RD Rio M. tuberculosis.
Background: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiologi... more Background: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages.
Uploads
Papers by Maranibia Oelemann