Activity of rifampicin and linezolid combination in Mycobacterium tuberculosis.

BACKGROUND: Linezolid (LZD) is not commonly used for treating tuberculosis (TB), but in some patients with drug-resistant TB it is being used. However, the in vitro LZD activity, in combination with rifampicin (RIF) against Mycobacterium tuberculosis has not been fully elucidated. AIMS: The aim of this study was to evaluate the in vitro activity of RIF/LZD combination against M. tuberculosis clinical isolates. MATERIALS AND METHODS: The activity of the RIF/LZD combination was firstly determined in M. tuberculosis H37Rv, 14 susceptible, 9 isoniazid nonresistant and 14 multi-drug resistant (MDR) M. tuberculosis clinical isolates by modified checkerboard assay, Resazurin Drugs Combination Microtiter Assay (REDCA). After, the Time Kill Curve Assay, at 0.5 × MIC of drugs, in combination and alone, was performed in M. tuberculosis H37Rv and 8 (20.5%) of those clinical isolates, which the RIF/LZD combination showed to have synergistic effect by the checkerboard assay. RESULTS AND CONCLUSION: By Time Kill Curve Assay, we could observe in M. tuberculosis H37Rv and susceptible isolates, that LZD alone, at sub inhibitory concentration, has poor effect on the bacillus death. In some cases, the bacillus growth stayed constant while in others showed regrowth at the eighth day of drug exposure. RIF alone exhibits potent concentration-dependent bactericidal activity, and was strongly dependent by the drug exposure time. The RIF/LZD combination accomplished a bacteriostatic effect in the reference strain and susceptible isolates. For the RIF resistant isolates, the RIF/LZD combination did not enhance the effect in killing bacillus. In this sense, additional, in vitro and in vivo studies are needed to evaluate the effect of RIF/LZD combination in order to better understand the adjunctive action of LZD in the treatment of TB and prevent the emergence of mutants with resistance to the available anti-TB drugs.

Activity of rifampicin and linezolid combination in Mycobacteriumtuberculosis

Background: Linezolid (LZD) is not commonly used for treating tuberculosis (TB), but in some patients with drug-resistant TB it is being used. However, the in vitro LZD activity, in combination with rifampicin (RIF) against Mycobacterium tuberculosis has not been fully elucidated.Aims: The aim of this study was to evaluate the in vitro activity of RIF/LZD combination against M. tuberculosis clinical isolates.Materials and methods: The activity of the RIF/LZD combination was firstly determined in M. tuberculosis H37Rv, 14 susceptible, 9 isoniazid nonresistant and 14 multi-drug resistant (MDR) M. tuberculosis clinical isolates by modified checkerboard assay, Resazurin Drugs Combination Microtiter Assay (REDCA). After, the Time Kill Curve Assay, at 0.5 MIC of drugs, in combination and alone, was performed in M. tuberculosis H37Rv and 8 (20.5%) of those clinical isolates, which the RIF/LZD combination showed to have synergistic effect by the checkerboard assay.Results and conclusion: By Time Kill Curve Assay, we could observe in M. tuberculosis H37Rv and susceptible isolates, that LZD alone, at sub inhibitory concentration, has poor effect on the bacillus death. In some cases, the bacillus growth stayed constant while in others showed regrowth at the eighth day ofdrug exposure. RIF alone exhibits potent concentration-dependent bactericidal activity, and was strongly dependent by the drug exposure time. The RIF/LZD combination accomplished a bacteriostatic effect in the reference strain and susceptible isolates. For the RIF resistant isolates, the RIF/LZD combination did not enhance the effect in killing bacillus. In this sense, additional, in vitro and in vivo studies are needed to evaluate the effect of RIF/LZD combination in order to better understand the adjunctive action of LZD in the treatment of TB and prevent the emergence of mutants with resistance to the available anti-TB drugs.

Critical analysis: use of polymerase chain reaction to diagnose leprosy

ABSTRACT Leprosy is a neglected tropical disease and an important public health problem, especially in developing countries. It is a chronic infectious disease that is caused by Mycobacterium leprae, which has a predilection for the skin and peripheral nerves. Although it has low sensitivity, slit-skin smear (SSS) remains the conventional auxiliary laboratory technique for the clinical diagnosis of leprosy. Polymerase chain reaction (PCR) is a molecular biology technique that holds promise as a simple and sensitive diagnostic tool. In the present study, the performance of two PCR methods, using different targets, PCR-LP and PCR-P, were compared with SSS with regard to leprosy diagnosis in a reference laboratory. M. leprae DNA was extracted from 106 lymph samples of 40 patients who had clinical suspicion of leprosy. The samples were subjected to both PCR techniques and SSS. Amplification of the human b-globin gene was used as PCR inhibitor control. The specificity of both PCR techniques was 100%, and sensitivity was 0.007 and 0.015 µg/ml for PCR-LP and PCR-P, respectively. No significant difference was found between either the PCR-LP or PCR-P results and SSS results (p > 0.05). Although PCR is not yet a replacement for SSS in the diagnosis of leprosy, this technique may be used as an efficient auxiliary tool for early detection of the disease, especially in endemic regions. This strategy may also be useful in cases in which SSS results are negative (e.g., in paucibacillary patients) and cases in which skin biopsy cannot be performed.

RESUMO A hanseníase é uma doença tropical negligenciada e ainda um importante problema de saúde pública, especialmente nos países em desenvolvimento. É uma doença infecciosa crônica causada pelo Mycobacterium leprae, que tem predileção pela pele e nervos periféricos. Embora com baixa sensibilidade, o esfregaço de linfa (SSS) continua sendo o método laboratorial convencional auxiliar no diagnóstico clínico da hanseníase. A biologia molecular representada pela Reação em Cadeia da Polimerase (PCR) trouxe a expectativa de ser uma ferramenta diagnóstica simples e sensível. No presente estudo, o desempenho de dois métodos de PCR usando alvos diferentes, PCR-P e PCR-LP, foi comparado com SSS no diagnóstico da hanseníase em um laboratório de referência. DNA de M. leprae foi extraído de 106 amostras de linfa de 40 pacientes que apresentavam suspeita clínica de hanseníase. As amostras foram submetidas tanto a PCR como SSS. A amplificação do gene humano β-globina foi usada como controle de inibição da PCR. A especificidade de ambas as técnicas de PCR foi de 100% e a sensibilidade foi de 0,007 μg/mL e 0,015 μg/mL para a PCR-P e PCR-LP, respectivamente. Não se observou diferença estatística entre os resultados da PCR-LP e PCR-P, quando comparado com SSS (p > 0,05). Apesar de a PCR ainda não substituir o SSS no diagnóstico da hanseníase, esta técnica pode ser usada como ferramenta auxiliar eficiente para a detecção precoce da doença, especialmente em regiões endêmicas. Esta estratégia pode também ser útil nos casos em que os resultados de SSS forem negativos (ex. em pacientes paucibacilares) e em casos onde a biópsia da pele não pode ser realizada.

In vitro activity of rifampicin and verapamil combination in multidrug-resistant mycobacterium tuberculosis.

The aim of the present study was to evaluate the effect of the combination of rifampicin (RIF) and verapamil (VP) against the Mycobacterium tuberculosis H37Rv reference strain and six multidrug-resistant (MDR) M. tuberculosis clinical isolates by determining Time-Kill Curves and the ability to efflux drug by fluorometry. The RIF+VP combination showed synergism in one MDR clinical isolate. For the other five MDR clinical isolates, the drug combination showed no interaction. The MDR clinical isolate had lower ethidium bromide (EtBr) accumulation when exposed to the RIF+VP combination, compared with RIF and VP exposure alone. The other MDR clinical isolates showed no significant difference in EtBr accumulation. These results suggest greater efflux action in one of the MDR clinical isolates compared with the M. tuberculosis H37Rv reference strain. The other five MDR isolates may have additional mechanisms of drug resistance to RIF. The use of the RIF+VP combination made one MDR bacillus more susceptible to RIF probably by inhibiting efflux pumps, and this combination therapy, in some cases, may contribute to a reduction of resistance to RIF in M. tuberculosis.

Use of the Ogawa-Kudoh method to isolate mycobacteria in a tuberculosis reference laboratory in northwestern Paraná, Brazil

Culturing is the gold standard method for confirming a diagnosis of tuberculosis (TB). The Brazilian Ministry of Health recently proposed the use of the Ogawa-Kudoh method for sputa cultures to detect Mycobacterium tuberculosis. The aim of the present study was to evaluate 8 years of using the Ogawa-Kudoh method in a TB reference laboratory in northwestern Paraná, Brazil. The present study consisted of a retrospective analysis of sputa cultures records for the detection of mycobacteria using the Ogawa-Kudoh method in the Laboratory of Medical Bacteriology, Laboratory of Teaching and Research in Clinical Analysis (LEPAC), State University of Maringá, from July 2003 to September 2011. The following variables were analyzed: Ziehl Neelsen (Z-N) smears and cultures results and the age and gender of the patients. Sputa samples from 3,231 patients with suspected TB were analyzed. Of these, 67.17% were male with an average age of 45.58 years. Of the total number of Z-N-negative samples (n=2,949), 42 (1.42%) were positive for M. tuberculosis (p >0.05). The Ogawa-Kudoh method is an excellent tool for diagnosing pulmonary TB. It is easy to perform, requires less biosafety equipment than the Petroff method, has a low cost, and has good sensitivity for detecting of M. tuberculosis.

A cultura é o método padrão ouro para confirmação da tuberculose (TB). O Ministério da Saúde Brasileiro propôs, recentemente, a utilização do método de Ogawa-Kudoh para cultura de escarro na detecção de Mycobacterium tuberculosis. O objetivo deste estudo foi avaliar oito anos de utilização do método de Ogawa-Kudoh na rotina de um laboratório de referência na região noroeste do Paraná, Brasil. Realizou-se estudo retrospectivo dos registros das culturas de escarro para a detecção de micobactérias, usando o método Ogawa-Kudoh conduzido no Laboratório de Bacteriologia Médica, Laboratório de ensino e pesquisa em Análises Clínicas (LEPAC) da Universidade Estadual de Maringá (UEM), de Julho de 2003 a Setembro de 2011. As seguintes variáveis foram analisadas: esfregaço Ziehl Neelsen (Z-N), cultura, idade e sexo do paciente. Analisaram-se 3.231 amostras de escarro de pacientes com suspeita de tuberculose. Destes, 67,17% eram do sexo masculino com idade média de 45,58 anos. Do total de amostras Z-N negativas (n=2.949), 42 amostras (42/2949, 1,42%) apresentaram cultura positiva para M. tuberculosis (p>0,05). A utilização do método Ogawa-Kudoh representa excelente ferramenta para o diagnóstico precoce da TB pulmonar. É de fácil execução, requer menos equipamentos de biossegurança do que o método de Petroff, apresenta baixo custo e boa sensibilidade para detecção de M. tuberculosis.