Development of molecular markers of Mycobacterium tuberculosis rifampicin resistance gene rpoB by PARMS technology / 生物工程学报

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of / 生物医学与环境科学（英文）

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Nontuberculous mycobacteria in patients with suspected tuberculosis and the genetic diversity of Mycobacterium avium in the extreme south of Brazil / Micobactérias não tuberculosas em pacientes com suspeita de tuberculose e a diversidade genética de Mycobacterium avium no extremo sul do Brasil

ABSTRACT Objective: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. Methods: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. Results: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. Conclusions: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

RESUMO Objetivo: As micobactérias não tuberculosas (MNT) são um grupo heterogêneo de bactérias amplamente distribuídas na natureza e relacionadas com infecções oportunistas em seres humanos. Os objetivos deste estudo foram identificar MNT em pacientes com suspeita de tuberculose e culturas positivas e avaliar a diversidade genética de cepas identificadas como Mycobacterium avium. Métodos: Foram estudadas amostras pulmonares e extrapulmonares provenientes de 1.248 pacientes. As amostras que apresentaram resultado positivo em cultura e negativo para o complexo M. tuberculosis na identificação molecular foram avaliadas por meio da detecção dos genes hsp65 e rpoB e de sequenciamento de fragmentos conservados desses genes. Todas as cepas identificadas como M. avium foram genotipadas pelo método mycobacterial interspersed repetitive unit-variable-number tandem-repeat com oito loci. Resultados: Das 332 micobactérias isoladas, 25 (7,5%) eram MNT. Dessas 25, 18 (72%) eram M. avium, 5 (20%) eram M. abscessus, 1 (4%) era M. gastri e 1 (4%) era M. kansasii. As 18 cepas de M. avium apresentaram alta diversidade, e apenas duas eram geneticamente relacionadas. Conclusões: Esses resultados mostram a necessidade de considerar a investigação de MNT em pacientes com suspeita de tuberculose ativa e culturas positivas e de avaliar a diversidade genética de cepas de M. avium.

Detection of multidrug-resistant Mycobacterium tuberculosis strains isolated in Brazil using a multimarker genetic assay for katG and rpoB genes

Abstract Multidrug-resistant tuberculosis (MDRTB) is a serious world health problem that limits public actions to control tuberculosis, because the most used anti-tuberculosis first-line drugs fail to stop mycobacterium spread. Consequently, a quick detection through molecular diagnosis is essential to reduce morbidity and medical costs. Despite the availability of several molecular-based commercial-kits to diagnose multidrug-resistant tuberculosis, their diagnostic value might diverge worldwide since Mycobacterium tuberculosis genetic variability differs according to geographic location. Here, we studied the predictive value of four common mycobacterial mutations in strains isolated from endemic areas of Brazil. Mutations were found at the frequency of 41.9% for katG, 25.6% for inhA, and 69.8% for rpoB genes in multidrug-resistant strains. Multimarker analysis revealed that combination of only two mutations (“katG/S315T + rpoB/S531L”) was a better surrogate of multidrug-resistant tuberculosis than single-marker analysis (86% sensitivity vs. 62.8%). Prediction of multidrug-resistant tuberculosis was not improved by adding a third or fourth mutation in the model. Therefore, rather than using diagnostic kits detecting several mutations, we propose a simple dual-marker panel to detect multidrug-resistant tuberculosis, with 86% sensitivity and 100% specificity. In conclusion, this approach (previous genetic study + analysis of only prevalent markers) would considerably decrease the processing costs while retaining diagnostic accuracy.

Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data

BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.

Characterization of Francisella species isolated from the cooling water of an air conditioning system

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Report of rpoB mutation in clinically suspected cases of drug resistant leprosy: A study from Eastern India.

Background: The current strategy for leprosy control depends mainly on early case detection and providing the recommended multidrug therapy (MDT) dosage. Understanding the molecular mechanisms of drug resistance to each of these drugs is essential in providing effective treatment and preventing the spread of resistant strains in the community. The progress of molecular biology research provides a very effi cient opportunity for the diagnosis of drug resistance by in vitro method. Aim: We aimed to investigate the point mutations within the rpoB gene region of the Mycobacterium leprae genome, which are responsible for resistance to rifampicin, in order to determine the emergence of drug resistance in leprosy in the Kolkata region of West Bengal. Methods: A total of 50 patients with a relapse of leprosy were enrolled in the study. Skin smears were obtained for estimation of bacillary index and biopsies were obtained in 70% alcohol for extraction of DNA. The extracted DNA was amplifi ed by M. leprae-polymerase chain reaction (PCR) targeting rpoB gene region. Every single nucleotide base in the sequence is aligned to reference sequence and identity gaps were determined by NCBI – BLAST. Later in-silico analysis was done to identify the changes in the translated protein sequences. Results: A mutation at the base pair position 2275405 where G is replaced by C in the M. leprae genome, which corresponds to the coding region of rpoB gene (279 bp – 2275228 to2275506), was observed in two patients. This missense mutation in CAC codon brings about a glutamic acid to histidine change in the amino acid sequence of RNA polymerase beta subunit at the position 442 (Glu442His), a region specifi c for rifampicin interaction, which might be responsible for unresponsiveness to rifampicin by manifesting a stable bacteriological index in these 2 patients even after completion of 24 months of multibacillary multi-drug therapy (MB-MDT). Limitations: The major limitations of multipleprimer PCR amplifi cation refractory mutation system (MARS) assay is that it capable of detecting mutation at codon 425 and cannot distinguish any silent amino acid changes. Conclusion: The study indicates the existence of rifampicin drug resistance in Eastern India.

Molecular characterization of the first leptospires isolated from goats in Brazil

Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.

Determinants of emergency contraception non-use among women in unplanned or ambivalent pregnancies / Determinantes del no uso de la anticoncepción de emergencia entre mujeres con embarazo no planeado u ambivalente / Determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente

Objective To analyze the determinants of emergency contraception non-use among women in unplanned and ambivalent pregnancies. Method Cross-sectional study with a probabilistic sample of 366 pregnant women from 12 primary health care units in the city of São Paulo, Brazil. A multinomial logistic regression was performed, comparing three groups: women who used emergency contraception to prevent ongoing pregnancies (reference); women who made no use of emergency contraception, but used other contraceptive methods; and women who made no use of any contraceptive methods at all. Results Cohabitation with a partner was the common determinant of emergency contraception non-use. No pregnancy risk awareness, ambivalent pregnancies and no previous use of emergency contraception also contributed to emergency contraception non-use. Conclusion Apart from what is pointed out in the literature, knowledge of emergency contraception and the fertile period were not associated to its use. .

Objetivo Analizar los determinantes del no uso de la anticoncepción de emergencia entre las mujeres con embarazo no planeado o ambivalente. Método Estudio transversal en una muestra probabilística de 366 mujeres embarazadas de 12 Unidades Básicas de Salud de São Paulo. Mediante regresión logística multinomial, se comparó tres grupos de mujeres: aquellas que usaron la anticoncepción de emergencia para prevenir el embarazo en curso (referencia), aquellas que usaron algún método anticonceptivo, pero no la anticoncepción de emergência; y aquellas que no usaron ningún método. Resultados Los hallazgos mostraron que vivir com la pareja fue el determinante común del no uso de la anticoncepción de emergencia. No tener conciencia del riesgo de embarazo, estar en un embarazo ambivalente y nunca tener utilizado la anticoncepción de emergencia también fueron associados con su no uso para prevenir el embarazo en curso. Conclusión Contrariamente a lo que reporta la literatura, el conocimiento de la anticoncepción de emergencia y el período fértil no mostró asociación con el no uso. .

Objetivo Analisar os determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente. Método Estudo transversal com amostra probabilística de 366 gestantes de 12 Unidades Básicas de Saúde da cidade de São Paulo. Por meio de regressão logística multinomial, compararam-se três grupos de mulheres: as que usaram anticoncepção de emergência para prevenir a gravidez em curso (referência); as que usaram algum método contraceptivo, mas não anticoncepção de emergência; e as que não usaram nenhum método. Resultados Os achados mostraram que morar com o parceiro foi o determinante comum do não uso da anticoncepção de emergência. Não ter consciência do risco de engravidar, estar em uma gravidez ambivalente e nunca ter usado anticoncepção de emergência também foram associados ao seu não uso para prevenir a gravidez em curso. Conclusão Diferentemente do que relata a literatura, o conhecimento sobre anticoncepção de emergência e sobre o período fértil não mostrou qualquer associação ao não uso. .

Expression of rna polymerase IV and V in oryza sativa

RNA polymerase IV and V are principal players in the RdDM pathway, where their current study has shown interaction of several factors that control DNA silencing of intergenic regions and siRNA production. DNA silencing is an important process during cell differentiation, nuclear structure and viral control. However, RNA pol IV and V are yet to be study in model monocot systems like Oryza sativa that can provide further information on genetic silence mechanism in plats. We show the expression pattern of these polymerases in tissues extracts of Oryza sativa. Detectable amounts of these polymerases are found in specific adult plant tissues and particularly expressed during somatic embryogenesis but not during early stages of normal embryo development. The use of synthetic auxin leads to an induction of both RNA pol IV and V in scutellum tissue where nuclear localization may be required for genome reorganization and gene silencing.

Diagnosis and Species Identification of Mycobacterial Infections by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of Sterile Body Fluids

BACKGROUND/AIMS: The development of effective, accurate, and rapid diagnostic methods for Mycobacterium infection and mycobacterial species identification is required. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is an easy, rapid and inexpensive technique for identifying Mycobacterium spp. METHODS: We performed PCR-RFLP to detect and identify Mycobacterium spp. from 10 sterile body fluids, including ascites, cerebrospinal fluid, pleural fluid, synovial fluid, and peritoneal dialysis fluid. Clinical samples were collected from patients with diagnoses of definite, probable or suspected mycobacterial infection. The conserved RNA polymerase genes of Mycobacterium spp. were amplified by PCR. RESULTS: The amplified 360-bp region of rpoB was digested with the restriction enzyme MspI or HaeIII. The PCRRFLP results for the clinical samples were identical to those for M. tuberculosis, M. fortuitum, M. intracellulare, and M. avium. In addition, the results of the PCR-RFLP were identical to those obtained by DNA sequencing. CONCLUSIONS: PCR-RFLP analysis of sterile body fluids may be a useful method for the diagnosis of mycobacterial infections and for the differentiation of mycobacterial species.

Correlation of mutations detected by INNO-LiPA with levels of rifampicin resistance in Mycobacterium tuberculosis.

BACKGROUND & OBJECTIVES: Due to emergence of drug resistance in Mycobacterium tuberculosis, there is a need to have accurate and rapid methods for detection of drug resistance to important drugs like rifampicin. The present study was aimed at evaluation of a commercially available INNO-LiPA assay, for the detection of mutation in rpoB gene region of M. tuberculosis and correlate these mutations with levels of rifampicin resistance for assessing their clinical relevance. METHODS: Fifty five well-characterized isolates of M. tuberculosis deposited from various regions of India in Mycobacterial Repository Centre at the CJILOMD, Agra were subjected to susceptibility testing for rifampicin at various concentrations of drug viz., 10, 40, 64, 128 microg/ml on Lowenstein- Jensen (LJ) medium. rpoB gene fragment (260 bp) was amplified using Rif-TB amplification kit and after hybridization, detection was done by using INNO-LiPA Rif TB kit. RESULTS: The rpoB gene could be amplified from DNA extracted from all the 55 culture isolates and showed clear hybridization pattern with M. tuberculosis complex specific probes on LiPA strips. Mutations detected were correlated with degree of rifampicin resistance. All the sensitive isolates (identified by MIC) were identified as rifampicin sensitive (100%) by INNO-LiPA as they exhibit positive for wild type 'S' probes and negative for 'R' probes. Two of the 5 isolates, resistant at 10 microg/ml and 40 microg/ml had either D516V, H526Y mutations or unknown mutations. Thirty (85.71%) isolates resistant at clinically relevant levels (64,128microg/ml) exhibited double, triple or more 'R' type mutations (R(2(D516V)), R(4a(H526Y)), R(4b(H526D)), R(5(S531L))) as well as unknown mutations present at 'S' probes region whereas remaining isolates did not show any mutation by this method. This method could identify with definitiveness 60 per cent ( 21/35) isolates as rifampicin resistant as mutations observed in others were also present in isolates with low levels of resistance. INTERPRETATION & CONCLUSION: The results indicate that INNO-LiPA Rif TB test is a rapid and easy to use method for detection of mutations associated with rifampicin resistance in M. tuberculosis. However, as some of these mutations are also present in isolates with low degree of resistance which are still microbiologically sensitive to rifampicin, there is a need to improve this assay by exclusion of some of the current probes and inclusion of more probes.

Rapid detection of mutations in rpoB gene of rifampicin resistant Mycobacterium tuberculosis strains by line probe assay.

BACKGROUND & OBJECTIVES: Multidrug resistant (MDR) tuberculosis (TB) is a problem of increasing importance in the world due to limited treatment options. Resistance to rifampicin results from nucleotide changes in the gene encoding the beta subunit of the RNA polymerase (rpoB) of Mycobacterium tuberculosis. Rifampicin resistance is considered as a marker for MDR TB. The nature and frequency of mutations in the rpoB gene of rifampicin resistant clinical isolates vary considerably according to the geographical location and very little information is available on specific mutational patterns in India. This study was undertaken to detect and characterize the rpoB gene mutation associated with rifampicin resistance in M. tuberculosis by line probe assay. METHODS: A total of 36 strains of M. tuberculosis were analysed by INNO-LiPA Rif TB and compared with the results of conventional susceptibility testing method. After PCR amplification of the region of RNA polymerase involved in rifampicin resistance, the amplified product was hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip. From the pattern obtained, the presence or absence of rifampicin resistance in the M. tuberculosis strains was assessed. RESULTS: It was found that the M. tuberculosis probe was 100 per cent specific; the most frequently observed mutation was His-526-Tyr in the rpoB gene; and correlation between the results of the LiPA and those obtained by the classical susceptibility testing was excellent (100%). INTERPRETATION & CONCLUSION: INNO LiPA was found to be a reliable, simple, rapid and informative tool for the early detection and characterization of rpoB mutation associated with rifampicin resistance in M. tuberculosis in the clinical laboratory setting and may constitute an important molecular method for the control of tuberculosis.

Prevalence of HIV-1 polymerase gene mutations in pre-treated patients in Thailand.

To determine the prevalence of drug resistance-conferring mutations in human immunodeficiency virus type 1 (HIV-1), 83 HIV-1 infected Thai patients who had been treated with any antiretroviral drug were studied. HIV-1 RNA was reverse transcribed and amplified by RT-PCR. The direct sequencing of HIV-1 reverse transcriptase (RT) and protease was then performed. Changes in nucleotide and amino acid sequences were determined by comparison with a pNL4-3 reference sequence. Data on mutations associated with resistance to antiretroviral drugs were obtained from literature. The mutations associated with lamivudine resistance (M184V/I) were found most often (in 45.7% of individuals). Zidovudine-resistant mutants: T215Y/F (36%), M41L (28%) and K70R (25.3%) were common; but mutations linked to didanosine (L74V) and multinucleoside-resistant genotypes (Q151M) were rarely recognized (2.4% and 3.6%, respectively). The stavudine-resistant mutant (V75T) and T69 insertions were not found. All subjects who had a significant exposure to antiretroviral drugs and current virological failure in the past carried drug-resistant genotypes. Genotypic resistance to zidovudine, lamivudine, zalcitabine, indinavir and ritonavir appeared in more than one third of the samples, which suggested that the prevalence of the HIV-1 resistance-conferring genotype resisting reverse transcriptase inhibitors and/or protease inhibitors was high in treatment experienced patients.

Genotypic subtyping of the HIV-1 polymerase gene in 30 naåve patients from Thailand.

To investigate the subtype classification of the circulating virus strains among infected Thai patients with human immunodeficiency virus type 1 (HIV-1). A random population of patients who were HIV-1 antibody positive after two independent screening assays was selected. HIV RNA from plasma samples was reverse-transcribed and amplified with specific primers that annealed to conserve regions of the HIV-1 pol gene. Amplified products were sequenced directly by using an automated sequencer. The sequencing products represent about 1.2 kb of the pol gene from each patient and they were phylogenetically analyzed and compared to the corresponding pol sequences of the published HIV-1 sequences of known genotypes. Genotype E was found in 25 of 30 patients (83.3%), and 5 patients (16.7%) were HIV-1 genotype B. The result confirmed that HIV-1 subtype E is still predominant in Thailand. Genotype B is found frequently, but there have been no examples of genotype A. In concordance with the serotypic assay, which was previously reported using the V3-peptide enzyme immunoassay (V3-PEIA), the genotypic assay of subtype E was high, at 80% and 83.3% in serotyping and genotyping, respectively. These findings of two subtypes with low heterogeneity indicate that Thailand may be a desirable site for evaluating candidate HIV-1 antiretroviral drugs and vaccines. The mixture of subtype E and B' strains also offers the opportunity to study phenotypic differences between the two subtypes.

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid