Moderate prevalence of HIV-1 transmitted drug resistance mutations in southern Brazil.

BACKGROUND: Despite the advances in therapy, the occurrence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a major obstacle to successful treatment. This study aimed to characterize the genetic diversity and to determine the prevalence of transmitted drug resistance mutations (TDRM) between individuals recently or chronically diagnosed with HIV-1 from Paraná, Brazil. METHODS: A total of 260 HIV-1 positive antiretroviral therapy-naïve patients were recruited to participate on the study, of which 39 were recently diagnosed. HIV-1 genotyping was performed using sequencing reaction followed by phylogenetic analyses to determine the HIV-1 subtype. TDRM were defined using the Calibrated Population Resistance Tool program. RESULTS: The HIV-1 subtypes frequency found in the studied population were 54.0% of subtype B, 26.7% subtype C, 6.7% subtype F1 and 12.7% recombinant forms. The overall prevalence of TDRM was 6.7%, including 13.3% for recently diagnosed subjects and 5.9% for the chronic group. CONCLUSIONS: The prevalence of resistance mutations found in this study is considered moderate, thus to perform genotyping tests before the initiation of antiretroviral therapy may be important to define the first line therapy and contribute for the improvement of regional prevention strategies for epidemic control.

Intramacrophage Mycobacterium tuberculosis efflux pump gene regulation after rifampicin and verapamil exposure.

Objectives: Since resistance of Mycobacterium tuberculosis (Mtb) partially derives from efflux pumps (EPs) in the plasma membrane, the current study evaluates EPs in Mtb exposed to rifampicin in the presence of the EP inhibitor verapamil, within a macrophage environment. Methods: Human acute monocytic leukaemia cell line THP-1 was infected with Mtb H37Rv and exposed to rifampicin and verapamil alone and in combination for 24 and 72 h. After RNA extraction, quantitative PCR was carried out for 11 EP genes using SYBR green PCR master mix in the StepOne™ Real-Time PCR System. Results: After 24 h of exposure to rifampicin, Mtb H37Rv showed that 10 EP genes were up-regulated when compared with the control. The rifampicin/verapamil combination induced down-regulation of 54.5% (6/11) of the EP genes. At 72 h, rifampicin exposure induced up-regulation of 10 EP genes and rifampicin/verapamil induced down-regulation of 8 EP genes, which suggests effective EP-inhibitory activity of verapamil against Mtb H37Rv in an intramacrophage environment. Conclusions: The current study demonstrated that rifampicin/verapamil caused down-regulation of several EP genes in Mtb inside the macrophage environment. In vivo trials may show that rifampicin/verapamil therapy could be of value in enhancing anti-TB treatment.

Promising Antituberculosis Activity of Piperine Combined with Antimicrobials: A Systematic Review.

Piperine, a bioactive compound from Piper nigrum and Piper longum, has shown promising activity as efflux pump (EP) inhibitor and as adjunct in treatment of tuberculosis (TB). The present systematic review investigated scientific studies of the activity of piperine against mycobacteria, with a focus on its mechanism of action, drug interactions, and antimycobacterial activity. A broad and rigorous literature search of three electronic databases (PubMed, Web of Knowledge, and LILACS) was performed according to the PRISMA statement. We considered studies that were published up to December 1, 2017. Google Scholar was also searched to increase the number of publications. We searched for articles using the search terms "piperine" and "Mycobacterium spp." The search yielded a total of 225 articles. After removing duplicate publications, 208 publications remained. Of these, we evaluated the full text of 13 articles. After applying the inclusion criteria, eight studies were included in the present systematic review. The results of the systematic review showed that piperine has promising anti-TB activity, mainly when combined with antimicrobials, and plays an important role as an EP inhibitor.

Morphological changes and differentially expressed efflux pump genes in Mycobacterium tuberculosis exposed to a rifampicin and verapamil combination.

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.

Detection of drug susceptibility in rapidly growing mycobacteria by resazurin broth microdilution assay.

The susceptibility of rapidly growing mycobacteria (RGM) to linezolid and ciprofloxacin was evaluated by using resazurin as a growth indicator. The assay with resazurin supplemented medium performed as well as its addition to the medium at reading time and was efficient for the determination for the susceptibility profile in RGM.

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.