No abstract

The aim of the present study was to (i) evaluate the in vitro action of rifampicin (RIF), ethambutol or isoniazid with efflux pumps inhibitors (EPIs) in Mycobacterium tuberculosis (Mtb) H37Rv and (ii) evaluate the morphological and efflux pumps (EPs) transcriptional changes by the action of rifampicin + verapamil combination (RIF + VP). The minimal inhibitory concentration and synergic effect of drug combinations were determined by Resazurin Microtiter Plate Assay and Resazurin Drugs Combination Microtiter Assay, respectively. VP showed greater capacity of ethidium bromide accumulation and RIF + VP had the lower fractional inhibitory concentration index. The RIF + VP exerted a similar reduction of viable cell counts to RIF by time-kill curve, but decreases in the expression of EPs genes were observed by Real time PCR at 72 h of RIF + VP exposure. Accumulative morphological changes (wrinkled and rounding) caused by each drug were observed by scanning electron microscopy after RIF + VP exposure. The downexpression of EPs related genes exposed to RIF + VP, suggest an effective inhibitory activity of VP in Mtb H37Rv. The role of EPs and the use of EPIs open up a powerful approach and the RIF + VP combination should be studied in Mtb more thoroughly.

By Time Kill Curve Assay, we could observe in M. tuberculosis HRv 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.

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.

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. 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 cas...