Resazurin Microtiter Assay for Clarithromycin Susceptibility Testing of Clinical Isolates of Mycobacterium abscessus Group.

BACKGROUND: Mycobacterium abscessus group has heterogeneous susceptibility pattern among species. The species is most common cause of nosocomial infections. Macrolides minimum Inhibitory concentration (MIC) determination is essential for the treatment. METHODS: Thirty-six strains were randomly selected for performing Resazurin Microtiter Assay (REMA) for clarithromycin testing in comparison to MIC test according to Clinical and Laboratory Standards Institute (2011) recommendation. REMA has been used for detection of drug resistance in M. tuberculosis. Extended incubation was performed to detect induced resistance. RESULTS: Thirty microliters of resazurin (0.01%) was added after visually taking MIC reading. Resistance was observed in 11.1% of M. bolletti and 4.8% of M. abscessus strains; and induced resistance was detected in 77.8% and 95.2% of M. bolletti and M. abscessus strains, respectively. All strains of M. massiliense were susceptible. The samples presented same MIC value both by visual reading and through resazurin. CONCLUSION: The present study showed 100% concordance between both readings, with REMA providing easier to read and report results benefit. This change in reading can also reflect on the MIC determination and report, improving the test.

Isolation and identification of environmental mycobacteria in the waters of a hemodialysis center.

The use of poorly treated water during hemodialysis may lead to contamination with nontuberculous mycobacteria (NTM). This study aimed to isolate and identify NTM species in the water of a Brazilian hemodialysis center. We collected 210 samples of water from the hydric system of the unit (post-osmosis system, hemodialysis rooms, reuse system, and hemodialysis equipment) and from the municipal supply network; we isolated the NTM by a classic microbiological technique and identified them by the PCR restriction enzyme pattern of the hsp65 gene (PRA). Fifty-one (24.3 %) of the collected samples tested positive for NTM; both the municipal supply network (2 samples, 3.2 %) and the hydric system of the hemodialysis center (49 samples, 96.1 %) contained NTM. We isolated and identified potentially pathogenic bacteria such as Mycobacterium lentiflavum (59.0 %) and M. kansasii (5.0 %), as well as rarely pathogenic bacteria like M. gordonae (24.0 %), M. gastri (8.0 %), and M. szulgai (4.0 %). The ability of NTM to cause diseases is well documented in the literature. Therefore, the identification of NTM in the water of a Brazilian hemodialysis center calls for more effective water disinfection procedures in this unit.

Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs.

We assessed the performance of REMA in comparison with BACTEC MGIT 960 in the susceptibility testing of 80 Mycobacterium tuberculosis clinical isolates from Clemente Ferreira Institute against four drugs. REMA proved to be a rapid and accurate method, providing excellent correlation with BACTEC MGIT 960, with the exception of results for the ethambutol drug.

Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay.

OBJECTIVES: The resazurin microtitre plate assay (REMA) was evaluated to determine the susceptibility of Mycobacterium tuberculosis to pyrazinamide, and was compared with the broth microdilution method (BMM), the absolute concentration method (ACM) and pyrazinamidase (PZase) determination. METHODS: Thirty-four M. tuberculosis clinical isolates (26 susceptible and 8 resistant to pyrazinamide) and reference strains M. tuberculosis H37Rv ATCC 27294 and Mycobacterium bovis AN5 were tested. RESULTS: REMA and BMM showed 100% specificity and sensitivity when compared with ACM; BMM, however, demanded more reading time. The PZase determination assay showed 87.50% and 100% sensitivity and specificity, respectively. CONCLUSIONS: All tested methods in this preliminary study showed excellent sensitivity and specificity for the determination of pyrazinamide susceptibility of M. tuberculosis, but REMA was faster, low-cost and easy to perform and interpret. Additional studies evaluating REMA for differentiating pyrazinamide-resistant and -susceptible M. tuberculosis should be conducted on an extended panel of clinical isolates.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis.

Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil. The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.

Mycobacterium tuberculosis population structure shift in a 5-year molecular epidemiology surveillance follow-up study in a low endemic agro-industrial setting in São Paulo, Brazil.

Starting with 257 outpatients attending the specialized health service for tuberculosis (TB) between 2002 and 2006 in Araraquara, an agro-industrial area with low tuberculosis (TB) incidence in São Paulo state, Brazil, positive mycobacterial cultures were obtained in 130 cases, of which 121 were confirmed as Mycobacterium tuberculosis complex. This report assesses the genetic diversity observed on 69.42% (n=84) of the clinical isolates, for which both spoligotyping and 12-loci MIRU typing data were fully interpretable. In order to monitor changes in the population dynamics of circulating M. tuberculosis strains over time, spoligotypes were compared from this study (n=84) with an earlier study from 1998 to 2001 (n=70 strains); and these two datasets from low-incidence Araraquara area were also compared with a 2-year cohort in the nearby higher-incidence São Paulo city area from 2006 to 2008 (n=93). The results obtained showed that with 58.3% (49/84) of the strains, the Latin-American-Mediterranean (LAM) was the predominant lineage in the present follow-up study; major patterns being SIT42/LAM9 11.9% (10/84), and SIT20/LAM1 10.7% (9/84). As compared with the 1998-2001 period when 40% (28/70) of the isolates belonged to the ill-defined T family, it was replaced by LAM strains between 2002 and 2006 with a visible shift to a population structure characteristic of the metropolitan São Paulo city. Further typing of the follow-up isolates from 2002 to 2006 using 12 loci MIRUs in conjunction with conventional epidemiology did not link this population structure shift to an increase in ongoing transmission or drug-resistance. Instead, it is most probably linked to movements of the important migrant community of Araraquara to higher TB incidence metropolitan areas such as São Paulo city. This is of particular concern owing to the increment in the global burden of LAM strains and the recent association of certain LAM sublineages with multidrug- and extensively drug-resistant TB. These observations suggest the need for further molecular monitoring of the TB population structure and the evaluation of transmission trends amongst migrant workers and other risk groups, such as persons in homeless shelters, in correctional facilities, drug users, and those with HIV infection, etc.

Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum.

INTRODUCTION: Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics. OBJECTIVE: This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi. METHODS: The phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification. RESULTS: Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (R(f)) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species. CONCLUSIONS: The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi.

Drug resistance in Mycobacterium tuberculosis clinical isolates from Brazil: phenotypic and genotypic methods.

We determined the susceptibility profile of 80 Mycobacterium tuberculosis (MTB) clinical isolates from Brazil against isoniazid (INH) and rifampicin (RIF) drugs by two phenotypic methods (Resazurin Microtiter Assay - REMA and BACTEC™ MGIT™ Mycobacterial Detection System). DNA polymorphisms were also determined by PCR-SSCP in isolates resistant to INH and RIF. BACTEC™ MGIT™ 960 detected 22 susceptible isolates to INH and RIF, 48 MDR isolates (resistant at least to INH and RIF) and nine mono-resistant isolates (eight to INH and one to RIF). REMA performance was determined by Receiver Operating Characteristic curve, whose assay was validated utilizing as reference the BACTEC™ MGIT™ 960 system. ROC curve showed cut-off values of 0.0625µg/mL and 0.125µg/mL, for INH and RIF, respectively. REMA-INH demonstrated sensitivity and specificity of 100% while REMA-RIF showed sensitivity of 97.2% and specificity of 100%. PCR-SSCP detected DNA polymorphisms in 87.5% and 75.5% of isolates classified as INH-resistant and RIF-resistant, respectively. One discordant sample found to RIF (resistant by BACTEC™ MGIT™ 960 and susceptible by REMA) showed no mutation by PCR-SSCP. In conclusion, our studies demonstrated that the combination of phenotypic method REMA, which allowed rapid detection of MDR-MTB with higher levels of sensitivity and specificity, with the genotypic method PCR-SSCP, which demonstrated high accuracy in the search of polymorphisms in the resistance genes, proved to be a useful strategy to study MDR-MTB clinical isolates from national reference center located in São Paulo city.

Topliss method in the optimization of salicylic Acid derivatives as potential antimycobacterial agents.

The Topliss method was used to guide a synthetic path in support of drug discovery efforts toward the identification of potent antimycobacterial agents. Salicylic acid and its derivatives, p-chloro, p-methoxy, and m-chlorosalicylic acid, exemplify a series of synthetic compounds whose minimum inhibitory concentrations for a strain of Mycobacterium were determined and compared to those of the reference drug, p-aminosalicylic acid. Several physicochemical descriptors (including Hammett's sigma constant, ionization constant, dipole moment, Hansch constant, calculated partition coefficient, Sterimol-L and -B4 and molecular volume) were considered to elucidate structure-activity relationships. Molecular electrostatic potential and molecular dipole moment maps were also calculated using the AM1 semi-empirical method. Among the new derivatives, m-chlorosalicylic acid showed the lowest minimum inhibitory concentration. The overall results suggest that both physicochemical properties and electronic features may influence the biological activity of this series of antimycobacterial agents and thus should be considered in designing new p-aminosalicylic acid analogs.

Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs

We assessed the performance of REMA in comparison with BACTEC MGIT 960 in the susceptibility testing of 80 Mycobacterium tuberculosis clinical isolates from Clemente Ferreira Institute against four drugs. REMA proved to be a rapid and accurate method, providing excellent correlation with BACTEC MGIT 960, with the exception of results for the ethambutol drug.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil.

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG), and within the inhA promoter and/or in structural gene. A small percentage (approximately 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh). Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum

INTRODUCTION: Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics. OBJECTIVE: This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi. METHODS: The phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification. RESULTS: Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (Rf) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959 bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species. CONCLUSIONS: The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis

Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil. The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG) , and within the inhA promoter and/or in structural gene. A small percentage (~ 10 percent) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh) . Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

Bacterial agents isolated from cerebrospinal fluid of patients with acquired immunodeficiency syndrome (AIDS) and neurological complications

Amostras de liquido cefalorraquidiano (LCR) de 2083 pacientes com Sindrome de Imunodeficiencia Adquirida (SIDA) e complicacoes neurologicas foram examinados durante um periodo de 7 anos (1984-1990). A porcentagem de pacientes que tiveram pelo menos um agente bacteriano cultivado do LCR foi de 6,2 por cento Mycobacterium tuberculosis foi o mais frequentemente isolado (4,3 por cento), seguido do complexo Mycobacterium avium ou MAC (0,7 por cento), de Pseudomas spp (0,5 por cento), Enterobacter spp (0,4 por cento), e Staphilococcus aureus (0,3 por cento). Entre 130 pacientes com cultura positiva, de 89 (68,5 por cento) foi isolado o M. tuberculosis e de 15 (11,6 por cento) MAC. A frequencia de isolamentos bacterianos foram mais frequentemente isolados de pacientes na faixa etaria de 21-30 anos e de mulheres (p<0.050).

Açäo in vitro de diferentes drogas, sobre o mycobacterium Kansasii / Mycobacterium Kansasii, drug susceptibility tests

A infecçäo causada pelo M.kansasii pode ser täo facilmente tratada como a tuberculose. Desde que a rifampicina foi introduzida nos esquemas terapêuticos a conversäo do escarro verifica-se em 100 por cento dos casos. O objetivo deste estudo foi contribuir no tratamento da micobacteriose causada pelo M.Kansasii. Através da determinaçäo da CIM, CBM e testes de sensibilidade pelo método das proporçöes, os autores comparam os resultados obtidos com os relatos da literatura internacional. Foram estudadas 30 cepas de M.Kansasii isoladas de pacientes com problemas pulmonares. Um perfil de sensibilidade das cepas frente à isoniazida, rifampicina, estreptomicina, etambutol, etionamida, kanamicina e ciprofloxacin foi realizado. A metodologia foi utilizada de acordo com as normas preconizadas para a bacteriologia das micobactérias. Os resultados obtidos mostraram sensibilidade do M.Kansasii à rifampicina, etionamida, kanamicina e ciprofloxacin, enquanto que para as demais drogas houve algumas variaçöes. Nossos dados mostram a importância da rifampicina nos esquemas terapêuticos, no tratamento inicial, e a utilidade dos testes de sensibilidade no retratamento

Atividade da violaceína in vitro sobre o Mycobacterium tuberculosis H37Ra / In vitro activity of violacein against Mycobacterium tuberculosis H37Ra

Este estudo relata a atividade antimicrobiana da violaceína, 3-[1,2-Dihidro-5 -(5-hidroxi-1H-indol-3-il)-2-oxo-3H-pirrol-3 ilideno] 1,3-dihidro-2H-indol-2-ona sobre a cepa padräo de M. tuberculosis H37Ra. A violaceína apresentou açäo antimicobacteriana in vitro com concentraçäo inibitória mínima (CIM) de 64 ug/mL e concentraçäo bactericida mínima (CBM) de 128 ug/mL. Estes valores säo comparáveis aos descritos na literatura para a pirazinamida

Microscopia de fluorescência como procedimento para a realizaçäo do exame baciloscópico em hanseníase / Fluorescent microscopy how procedure for the realization of the baciloscopic examination in leprosy

Visando comparar os métodos de microscopia fluorescente e de Ziehl-Neelsen para evidenciaçäo de bacilos álcool-ácido resistentes em material proveniente de derme de pacientes hansenianos, foram examinados 355 esfregaços. Obteve-se 98,81 por cento de concordância ajustada (KAPPA) entre os métodos. Verificou-se no entanto aproximadamente 55 por cento de resultados discordantes em relaçäo ao índice baciloscópico (Ridley), decorrentes de conatagens de bacilos por campo mais levadas pelo método fluorescente

Aspectos citológicos de amostras de escarro de pacientes com tuberculose pulmonar / Cytologic findings in sputum of patients with pulmonary tuberculosis

A tuberculose pulmonar é uma moléstia de ainda alta incidência em nosso país. Outras patologias podem estar associada a ela, como processos alérgicos pulmonares e parasitoses com estágio pulmonar em seu ciclo. a presença de eosinófilos no escarro de tuberculosos pode ser indicativa de uma dessas associaçöes. Este trabalho apresenta o estudo citológico de 74 esfregaços de escarro de pacientes sem tratamento, com diagnóstico inicial de tuberculose por baciloscopia e cultura. Células epitelióides e gigantes foram observadas em alguns esfregaços. A maioria deles mostrava fundo necrótico e substrato inflamatório intenso, com predomínio de linfócitos e/ou neutrófilos. Trinta e dois casos (43%) apresentavam quantidade significativa de eosinófilos, em contraste com a porcentagem de 20% de casos com eosinófilos no grupo controle (baciloscopia e cultura negativas). Esse achado sugere que os pacientes, além da tuberculose, podem apresentar outro processo patológico, que deve ser pesquisado

Perfil de resistência de Mycobacterium Tuberculosis no Estado de Säo Paulo, 1986 a 1990 / Resistence pattern of Mycobacterium tuberculosis from Säo Paulo State, Brazil, 1986 to 1990

Com o objetivo de determinar o perfil da resistência do Mycobacterium tuberculosis às drogas empregadas no tratamento da tuberculose no Estado de Säo Paulo, foram estudadas 1.668 cepas obtidas de amostra de escarro, sendo 254 doentes sem tratamento prévio e 1.414 na vigência de tratamento. A taxa de resistência inicial foi de 16,54//e a adquirida de 47,45//. A isoniazida apresentou resistência global de 10,62//e a rifampicina de 6,69//mostrando que as drogas mais importantes empregadas no tratamento da tuberculose estäo apresentando altas taxas de resistência. Este fato causa preocupaçäo pois pode implicar na falência do tratamento, acarretando sérias consequências para o controle da doença