Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.

BACKGROUND: To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE: To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD: 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS: With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION: Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil.

We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RD(Rio), and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C((-17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.

Tuberculosis recurrence in a high incidence setting for HIV and tuberculosis in Brazil.

BACKGROUND: To compare epidemiological data between recurrent cases after cure (RC), distinguishing relapse from reinfection, after dropout (RD) and new cases (NC) in an ambulatory setting in a TB-endemic country. METHODS: Records of patients who started treatment for pulmonary TB between 2004 and 2010 in a TB clinic were reviewed. Epidemiological data were analyzed. Spoligotyping and MIRU patterns were used to determine relapse or reinfection in 13 RC available. RESULTS: Of the eligible group (1449), 1060 were NC (73.2%), among the recurrent cases, 203 (14%) were RC and 186 (12.8%) were RD. Of RC, 171 (84.2%) occurred later than 6 months after a previous episode, 13 had available DNA, in 4 (30.7%) the disease was attributed to reinfection and in 9 (69.3%), to relapse. Comparing RC to NC, HIV (p < 0.0001) was independent risk factor for RC. When RC and RD were compared, alcohol abuse (p = 0.001) and treatment noncompliance (p = 0.006) were more frequent in RD. CONCLUSIONS: HIV is the sole more important associated factor for RC. This finding points the need to improve the approach to manage TB in order to decrease the chance for exposure especially in vulnerable people with increased risk of developing disease and to improve DOTS strategy to deal with factors associated to treatment noncompliance.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA.

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

Genetic Characterization and Population Structure of Drug-Resistant Mycobacterium tuberculosis Isolated from Brazilian Patients Using Whole-Genome Sequencing.

The present study aimed to determine the genetic diversity of isolates of Mycobacterium tuberculosis (Mtb) from presumed drug-resistant tuberculosis patients from several states of Brazil. The isolates had been submitted to conventional drug susceptibility testing for first- and second-line drugs. Multidrug-resistant (MDR-TB) (54.8%) was the most frequent phenotypic resistance profile, in addition to an important high frequency of pre-extensive resistance (p-XDR-TB) (9.2%). Using whole-genome sequencing (WGS), we characterized 298 Mtb isolates from Brazil. Besides the analysis of genotype distribution and possible correlations between molecular and clinical data, we determined the performance of an in-house WGS pipeline with other online pipelines for Mtb lineages and drug resistance profile definitions. Sub-lineage 4.3 (52%) was the most frequent genotype, and the genomic approach revealed a p-XDR-TB level of 22.5%. We detected twenty novel mutations in three resistance genes, and six of these were observed in eight phenotypically resistant isolates. A cluster analysis of 170 isolates showed that 43.5% of the TB patients belonged to 24 genomic clusters, suggesting considerable ongoing transmission of DR-TB, including two interstate transmissions. The in-house WGS pipeline showed the best overall performance in drug resistance prediction, presenting the best accuracy values for five of the nine drugs tested. Significant associations were observed between suffering from fatal disease and genotypic p-XDR-TB (p = 0.03) and either phenotypic (p = 0.006) or genotypic (p = 0.0007) ethambutol resistance. The use of WGS analysis improved our understanding of the population structure of MTBC in Brazil and the genetic and clinical data correlations and demonstrated its utility for surveillance efforts regarding the spread of DR-TB, hopefully helping to avoid the emergence of even more resistant strains and to reduce TB incidence and mortality rates.

Acetabularization as a Protective Factor in Rotator Cuff Arthropathy.

Objective To check if shoulders with acetabularization have better functional results in cases of rotator cuff arthropathy. Methods A clinical and radiological cross-sectional evaluation of 65 shoulders with rotator cuff arthropathy by measuring the range of motion (RoM) of the shoulder, the Constant-Murley score, and the radiological classifications of Hamada and Seebauer. The clinical findings were compared with the radiographic findings. Results According to the classification of Seebauer, we observed better results regarding the RoM in type-A shoulders. There was a statistically significant difference regarding anterior elevation and medial rotation between types A and B ( p < 0.05). Lateral rotation did not show a statistically significant difference between types A and B. The Constant-Murley score presented better results in type A, and there was a statistically significant difference between groups A and B ( p < 0.05). According to the classification of Hamada, we observed that the RoM had better results in types 3, 2 and 1, and there was a statistically significant difference regarding anterior elevation and medial rotation ( p < 0.05) compared with groups 4A, 4B and 5. There was no statistically significant difference between the Hamada groups regarding lateral rotation. According to Hamada, the Constant-Murley score showed better results in types 3, 1 and 2, and there was a statistically significant difference between groups 3 and 5. Conclusion The RoM and shoulder function were better in patients with acetabularization (Seebauer 1A and Hamada 3), and worse in those with glenohumeral arthrosis (Seebauer 1B, 2B and Hamada 4A, 4B and 5).

A highly rifampicin resistant Mycobacterium tuberculosis strain emerging in Southern Brazil.

Here we described phenotypical, molecular and epidemiological features of a highly rifampicin-resistant Mycobacterium tuberculosis strain emerging in Southern Brazil, that carries an uncommon insertion of 12 nucleotides at the codon 435 in the rpoB gene. Employing a whole-genome sequencing-based study on drug-resistant Mycobacterium tuberculosis strains, we identified this emergent strain in 16 (9.19%) from 174 rifampicin-resistant clinical strains, all of them belonging to LAM RD115 sublineage. Nine of these 16 strains were available to minimum inhibitory concentration determination and for all of them was found a high rifampicin-resistance level (≥to 32 mg/L). This high resistance level could be explained by structural changes into the RIF binding site of RNA polymerase caused by the insertions, and consequent low-affinity interaction with rifampicin complex confirmed through protein modeling and molecular docking simulations. Epidemiological investigation showed that most of the individuals (56.25%) infected by the studied strains were prison inmate individuals or that spent some time in prison. The phylogenomic approach revealed that strains carrying on insertion belonged to same genomic cluster, evidencing a communal transmission chain involving inmate individuals and community. We stress the importance of tuberculosis genomic surveillance and introduction of measures to interrupt Mycobacterium tuberculosis transmission chain in this region.

Clonal expansion across the seas as seen through CPLP-TB database: A joint effort in cataloguing Mycobacterium tuberculosis genetic diversity in Portuguese-speaking countries.

Tuberculosis (TB) remains a major health problem within the Community of Portuguese Language Speaking Countries (CPLP). Despite the marked variation in TB incidence across its member-states and continued human migratory flux between countries, a considerable gap in the knowledge on the Mycobacterium tuberculosis population structure and strain circulation between the countries still exists. To address this, we have assembled and analysed the largest CPLP M. tuberculosis molecular and drug susceptibility dataset, comprised by a total of 1447 clinical isolates, including 423 multidrug-resistant isolates, from five CPLP countries. The data herein presented reinforces Latin American and Mediterranean (LAM) strains as the hallmark of M. tuberculosis populational structure in the CPLP coupled with country-specific differential prevalence of minor clades. Moreover, using high-resolution typing by 24-loci MIRU-VNTR, six cross-border genetic clusters were detected, thus supporting recent clonal expansion across the Lusophone space. To make this data available to the scientific community and public health authorities we developed CPLP-TB (available at http://cplp-tb.ff.ulisboa.pt), an online database coupled with web-based tools for exploratory data analysis. As a public health tool, it is expected to contribute to improved knowledge on the M. tuberculosis population structure and strain circulation within the CPLP, thus supporting the risk assessment of strain-specific trends.

Genetic diversity of Mycobacterium tuberculosis isoniazid monoresistant and multidrug-resistant in Rio Grande do Sul, a tuberculosis high-burden state in Brazil.

Tuberculosis (TB) remains a major public health problem in the world and Brazil is among the countries with the highest incidence and prevalence rates, and Rio Grande do Sul, a Brazilian state, occupy a prominent position. Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) further aggravates this scenario, making it more difficult to treat and control the disease. Isoniazid monoresistance (IMR) may increase the risk of progression to MDR-TB and treatment failure. However, most drug resistance molecular tests only focus on detecting rifampicin (RIF) resistance.In the present study, we characterized a total of 63 drug resistant isolates of M. tuberculosis (35 MDR, 26 IMR and two isolates monoresistant to rifampicin [RMR]) of the Rio Grande do Sul state by MIRU-VNTR (24 loci), spoligotyping, presence of RDRio, fbpC103, pks15/1 and sequencing of the katG, rpoB and inhA genes. We observed a higher proportion of the LAM family 30/63 (47.61%). In IMR, mutations were found in the katG gene (98% at codon 315) in 72.5%, and mutations in the promoter region of the inhA gene in 6.25% of the isolates. In MDR-TB and RMR-TB isolates, 92.1% had mutations in the rpoB gene (57% at codon 531). The presence of a 12 bp insertion between codons 516 and 517 of the rpoB gene in MDR-TB isolates was found in five isolates. In conclusion, we observed that the highest frequency of IMR-TB and MDR-TB strains belong to the LAM and Haarlem genotypes in Rio Grande do Sul state. A significant number of isolates previously characterized as Mycobacterium pinnipedi2 through spoligotyping were found to belong to the M. tuberculosis LAM family. This was responsible for a number of significant cases and the molecular profile of this strain and the pattern of mutations related to drug resistance were analyzed. These findings may contribute to a better understanding about the spread of M. tuberculosis resistant in southern of Brazil.

Tendon of the long head of the biceps originating from the rotator cuff - An uncommon anatomical variation: case report.

Anatomical variations at the origin of the biceps tendon have been described by several authors, but occurrences of an origin in the supraspinatus are rare. It is unclear whether this variation might contribute toward pathological conditions of the shoulder. Our objective here was to describe a case of an anatomical variation in the origin of the tendon of the long head of the biceps. The clinical information, preoperative images and arthroscopic images relating to a patient with an aberrant origin of the long head of the biceps, which was observed during shoulder arthroscopy, were reviewed. In this case study, the origin of the biceps was found in the rotator cuff, without any origin from the supraglenoid tubercle or upper labrum. This variant did not seem to contribute toward the pathological condition of the shoulder, and standard treatment for the concomitant condition was sufficient for treating it.

As variações anatômicas na origem do tendão do bíceps foram descritas por vários autores, mas a ocorrência de sua origem no supraespinhal é rara. Não está claro se essa variação pode contribuir para condições patológicas do ombro. Nosso objetivo é descrever um caso de uma variação anatômica da origem da cabeça longa do tendão do bíceps.Informações clínicas, imagens pré-operatórias e imagens artroscópicas foram revisadas a partir de um paciente que teve uma origem aberrante da cabeça longa do bíceps observada durante a artroscopia do ombro.Neste estudo de caso, a origem do bíceps foi encontrada no manguito rotador, sem origem do tubérculo supraglenoidal ou labrum superior. Essa variante não parece contribuir para a patologia ombro e o tratamento padrão de patologia concomitante foi suficiente.

Acetabularization as a Protective Factor in Rotator Cuff Arthropathy / A acetabularização como fator protetor na artropatia do manguito rotador

Abstract Objective To check if shoulders with acetabularization have better functional results in cases of rotator cuff arthropathy. Methods A clinical and radiological cross-sectional evaluation of 65 shoulders with rotator cuff arthropathy by measuring the range of motion (RoM) of the shoulder, the Constant-Murley score, and the radiological classifications of Hamada and Seebauer. The clinical findings were compared with the radiographic findings. Results According to the classification of Seebauer, we observed better results regarding the RoM in type-A shoulders. There was a statistically significant difference regarding anterior elevation and medial rotation between types A and B (p< 0.05). Lateral rotation did not show a statistically significant difference between types A and B. The Constant-Murley score presented better results in type A, and there was a statistically significant difference between groups A and B (p< 0.05). According to the classification of Hamada, we observed that the RoM had better results in types 3, 2 and 1, and there was a statistically significant difference regarding anterior elevation and medial rotation (p< 0.05) compared with groups 4A, 4B and 5. There was no statistically significant difference between the Hamada groups regarding lateral rotation. According to Hamada, the Constant-Murley score showed better results in types 3, 1 and 2, and there was a statistically significant difference between groups 3 and 5. Conclusion The RoM and shoulder function were better in patients with acetabularization (Seebauer 1A and Hamada 3), and worse in those with glenohumeral arthrosis (Seebauer 1B, 2B and Hamada 4A, 4B and 5).

Resumo Objetivo Verificar se os ombros com acetabularização têm melhores resultados funcionais nos casos de artropatia do manguito rotador. Métodos Avaliação transversal clínica e radiológica de 65 ombros com artropatia do manguito rotador por meio da mensuração da amplitude de movimento (ADM) do ombro, do escore de Constant-Murley, e das classificações radiológicas de Hamada e Seebauer. Os achados clínicos foram comparados com os radiográficos. Resultados Segundo a classificação de Seebauer, com relação à ADM, observamos melhores resultados nos tipos A. Houve diferença estatística significativa na elevação anterior, e rotação medial entre os tipos A e B (p< 0.05). A rotação lateral não demonstrou diferença estatística significativa entre os tipos A e B. O escore de Constant-Murley apresentou melhores resultados nos tipos A, e houve diferença estatística significativa entre os grupos A e B (p< 0,05). Segundo a classificação de Hamada, observamos que a ADM teve melhores resultados nos tipos 3, 2 e 1, e houve diferença estatística significativa para a elevação anterior e a rotação medial (p< 0,05) quando comparadas com os grupos 4A, 4B e 5. Não houve diferença estatística significativa entre os grupos de Hamada em relação à rotação lateral. Ainda segundo Hamada, o escore de Constant-Murley apresentou melhores resultados nos tipos 3, 1 e 2, e houve diferença estatística significativa entre os grupos 3 e 5. Conclusão A ADM e a função do ombro apresentavam-se melhores nos pacientes com acetabularização (Seebauer 1A e Hamada 3), e piores naqueles com artrose glenoumeral (Seebauer 1B, 2B e Hamada 4A, 4B e 5).

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Tendon of the long head of the biceps originating from the rotator cuff - An uncommon anatomical variation: case report / Tendão da cabeça longa do bíceps originado do manguito rotador - Uma variação anatômica incomum: relato de caso

Anatomical variations at the origin of the biceps tendon have been described by several authors, but occurrences of an origin in the supraspinatus are rare. It is unclear whether this variation might contribute toward pathological conditions of the shoulder. Our objective here was to describe a case of an anatomical variation in the origin of the tendon of the long head of the biceps. The clinical information, preoperative images and arthroscopic images relating to a patient with an aberrant origin of the long head of the biceps, which was observed during shoulder arthroscopy, were reviewed. In this case study, the origin of the biceps was found in the rotator cuff, without any origin from the supraglenoid tubercle or upper labrum. This variant did not seem to contribute toward the pathological condition of the shoulder, and standard treatment for the concomitant condition was sufficient for treating it.

As variações anatômicas na origem do tendão do bíceps foram descritas por vários autores, mas a ocorrência de sua origem no supraespinhal é rara. Não está claro se essa variação pode contribuir para condições patológicas do ombro. Nosso objetivo é descrever um caso de uma variação anatômica da origem da cabeça longa do tendão do bíceps. Informações clínicas, imagens pré-operatórias e imagens artroscópicas foram revisadas a partir de um paciente que teve uma origem aberrante da cabeça longa do bíceps observada durante a artroscopia do ombro. Neste estudo de caso, a origem do bíceps foi encontrada no manguito rotador, sem origem do tubérculo supraglenoidal ou labrum superior. Essa variante não parece contribuir para a patologia ombro e o tratamento padrão de patologia concomitante foi suficiente.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

Multi-Locus sequence typing of Treponema pallidum in male patients with genital ulcers in a public STD clinic in South Brazil: a new allele and almost complete macrolide resistance / Tipificación de Treponema pallidum mediante MLST en pacientes masculinos con úlceras genitales en una clínica pública de ETS en el sur de Brasil: un nuevo alelo y resistencia casi completa a los macrólidos. / Tipagem do Treponema pallidum por MLST em pacientes do sexo masculino com úlceras genitais em uma clínica pública de DST no Sul do Brasil: um novo alelo e resistência quase completa aos macrolídeos

Objective: Considering the lack of data on T. pallidum genotyping in Brazil, we aimed to study its strains and their resistance to macrolides in genital ulcers suggestive of syphilis.Methods: Men with genital ulcers suggestive of syphilis were invited to participate. Samples were collected with a dry cotton swab and immersed in a 0.9% NaCl solution. Detection was done by PCR amplification of 260 bp of the tpp47 gene. The PCR product was analyzed by electrophoresis in a 2% agarose gel containing 0.05% ethidium bromide. Positive PCR samples were analyzed by MLST (sequencing of chromosomal loci TP0136, TP0548, and TP0705). The A2058G and A2059G mutations in the 23S rRNA gene were evaluated by nested PCR. DNA sequencing was analyzed using Bioedit software (Tom Hall, USA). Genotyping was performed using the PubMLST online platform (Grillová scheme). Results: All subjects were residents of Porto Alegre and aged between 19 and 66 years. Of the 43 samples, 32 were positive for PCR for T. pallidum. Thirty strains were available for genotyping and belonged to the SS14-like (73.3%) or Nicholslike (20%) Clonal Complex. Three complete MLST profiles were identified (1.3.1; 9.7.3 and 28.7.3), and a new allele was identified in one sample (approved by pubMLST curators as TP0705-22). Only one sample did not present the 2058 mutation in the 23S rRNA gene.Conclusion: Our study identified genetic diversity in T. pallidum DNA using MLST with allelic variants for TP0136, TP0548, and TP0705, including a new allele. A single sample was characterized as genotypically susceptible to macrolides. All other samples (more than 95%) presented the A2058G mutation in the 23S rRNA gene, which causes resistance to macrolides. Improving understanding of the local epidemiology of T. pallidum with representative samples that allow cofactor analysis is crucial for prevention and care.

Objetivo: Considerando la falta de datos sobre el genotipado de T. pallidum en Brasil, nos propusimos estudiar sus cepas y su resistencia a macrólidos en úlceras genitales sugestivas de sífilis. Métodos: Se invitó a participar a hombres con úlceras genitales sugestivas de sífilis. Las muestras se recogieron con un hisopo de algodón seco y se sumergieron en una solución de NaCl al 0,9%. La detección se realizó mediante amplificación por PCR de 260 pb del gen tpp47. El producto de la PCR se analizó mediante electroforesis en un gel de agarosa al 2% que contenía bromuro de etidio al 0,05%. Las muestras de PCR positivas se analizaron mediante MLST (secuenciación de los loci cromosómicos TP0136, TP0548 y TP0705). Las mutaciones A2058G y A2059G en el gen 23S rRNA se evaluaron mediante PCR anidada. La secuenciación de ADN se analizó utilizando el software Bioedit (Tom Hall, EE. UU.). El genotipado se realizó utilizando la plataforma en línea PubMLST (esquema Grillová). Resultados: Todos los sujetos eran residentes de Porto Alegre y tenían edades comprendidas entre 19 y 66 años. De las 43 muestras, 32 resultaron positivas a la PCR para T. pallidum. Treinta cepas estaban disponibles para el genotipado y pertenecían al Complejo Clonal tipo SS14 (73,3%) o tipo Nichols (20%). Se identificaron tres perfiles MLST completos (1.3.1; 9.7.3 y 28.7.3) y se identificó un nuevo alelo en una muestra (aprobado por los curadores de pubMLST como TP0705-22). Sólo una muestra no presentó la mutación 2058 en el gen 23S rRNA. Conclusión: Nuestro estudio identificó diversidad genética en el ADN de T. pallidum utilizando MLST con variantes alélicas para TP0136, TP0548 y TP0705, incluido un nuevo alelo. Una sola muestra se caracterizó como genotípicamente susceptible a macrólidos. El resto de muestras (más del 95%) presentaron la mutación A2058G en el gen 23S rRNA, que provoca resistencia a los macrólidos. Mejorar la comprensión de la epidemiología local de T. pallidum con muestras representativas que permitan el análisis de cofactores es crucial para la prevención y la atención.

Objetivo: Considerando a ausência de dados sobre genotipagem de T. pallidum no Brasil, objetivamos estudar suas cepas e sua resistência aos macrolídeos em úlceras genitais sugestivas de sífilis.Métodos: Foram convidados a participar homens com úlceras genitais sugestivas de sífilis. As amostras foram coletadas com swab de algodão seco e imersas em solução de NaCl 0,9%. A detecção foi feita por amplificação por PCR de 260 pb do gene tpp47. O produto de PCR foi analisado por eletroforese em gel de agarose a 2% contendo brometo de etídio a 0,05%. Amostras de PCR positivas foram analisadas por MLST (sequenciamento dos loci cromossômicos TP0136, TP0548 e TP0705). As mutações A2058G e A2059G no gene 23S rRNA foram avaliadas por nested PCR. O sequenciamento de DNA foi analisado utilizando o software Bioedit (Tom Hall, EUA). A genotipagem foi realizada utilizando a plataforma online PubMLST (esquema Grillová).Resultados: Todos os sujeitos eram residentes de Porto Alegre e tinham idade entre 19 e 66 anos. Das 43 amostras, 32 foram positivas para PCR para T. pallidum. Trinta cepas estavam disponíveis para genotipagem e pertenciam ao Complexo Clonal SS14-like (73,3%) ou Nichols-like (20%). Três perfis completos de MLST foram identificados (1.3.1; 9.7.3 e 28.7.3), e um novo alelo foi identificado em uma amostra (aprovado pelos curadores do pubMLST como TP0705-22). Apenas uma amostra não apresentava a mutação 2058 no gene 23S rRNA.Conclusão: Nosso estudo identificou diversidade genética no DNA de T. pallidum usando MLST com variantes alélicas para TP0136, TP0548 e TP0705, incluindo um novo alelo. Uma única amostra foi caracterizada como genotípicamente suscetível a macrolídeos. Todas as demais amostras (mais de 95%) apresentaram a mutação A2058G no gene 23S rRNA, que causa resistência aos macrolídeos. Melhorar a compreensão da epidemiologia local do T. pallidum com amostragens representativas que permitam análise de cofatores é crucial para a prevenção e o cuidado.