Evaluation of the Ogawa-Kudoh method for tuberculosis isolation in two health units in Mozambique.

BACKGROUND: Mozambique is among the highest tuberculosis, tuberculosis-HIV and multidrug-resistant-tuberculosis burden countries. Although molecular technologies are available in-country, mycobacterial isolation through culture remains an important tool for tuberculosis diagnostics and drug susceptibility testing. OBJECTIVE: We evaluated the use of the Ogawa-Kudoh (OK) mycobacterial culture, a simple technique, to isolate Mycobacterium tuberculosis in two health units, in Maputo City, Mozambique. METHODS: From May to December 2014, 122 patient samples were collected in Chamanculo General Hospital and Polana Caniço General Hospital. The specimens were first tested in the health units using the OK method and afterwards shipped to the National Tuberculosis Reference Laboratory for mycobacterial culture using the NALC-NaOH-Citrate (NALC) decontamination method followed by inoculation in Lowenstein Jensen (LJ) solid media as the reference standard. RESULTS: Among 107 samples with valid results, 98 (91.6%) had concordant results in both methods; 9 (8.4%) had discordant results. The contamination rate was 4.1% (5/122) for the OK and 9.0% (11/122) for the NALC/LJ methods. The sensitivity of OK was 80% (95% confident interval [CI]: 51.4-94.7) and the specificity was 94% (95% CI: 85.8-97.3). The degree of agreement between both methods was moderate (Kappa: 0.68; 95% CI: 0.48-0.89). CONCLUSION: The OK method showed satisfactory sensitivity and specificity. The method also had a lower contamination rate when compared to the NALC/LJ. Similar to other studies in resource-limited settings, our findings showed that the OK method can effectively be implemented in settings with limited laboratory capacity to isolate tuberculosis bacteria by culture for further testing.

Genetic correlates of clarithromycin susceptibility among isolates of the Mycobacterium abscessus group and the potential clinical applicability of a PCR-based analysis of erm(41).

Objectives: To define the genetic basis of clarithromycin resistance among isolates of the Mycobacterium abscessus group (MAG). Methods: We analysed 133 isolates identified as MAG. Species identification was confirmed by sequencing the rpoB gene. Clarithromycin susceptibility testing was performed according to CLSI recommendations, with an extended 14 day incubation. Known resistance genotypes of erm(41) and rrl were identified by sequencing; the presence of deletions in erm(41) was detected by PCR. Results: The 133 MAG isolates included 82 M. abscessus, 27 Mycobacterium massiliense and 24 Mycobacterium bolletii. After the 3 day incubation, only five isolates demonstrated clarithromycin resistance (R); after 14 days of extended incubation, an additional 92 exhibited inducible resistance (IR), with the remaining being susceptible (S). The distribution of susceptibility phenotypes varied among the species. Among M. abscessus isolates, 11% were S, 84% IR and 5% R; among M. bolletii isolates, 96% were IR and 4% R; and among M. massiliense isolates 100% were S. Sequencing of rrl identified only a single isolate with the A2058G mutation. Deletions in erm(41) were present in 30 susceptible isolates; among the remaining 103 isolates, 97 were R or IR (sensitivity, 83%; specificity, 100%; positive predictive value, 100%; negative predictive value, 94%). Among the six susceptible isolates without deletions, all carried the erm(41) T28C point mutation. Conclusions: A significant proportion of MAG isolates demonstrate inducible resistance to clarithromycin that is only detectable with an extended 14 day incubation. Further, the majority of clarithromycin-susceptible MAG isolates have characteristic deletions in erm(41) that can rapidly and reliably be detected by a simple PCR.

Paepalanthus spp: antimycobacterial activity of extracts, methoxylated flavonoids and naphthopyranone fractions

Paepalanthus spp., Eriocaulaceae, are native plants from Brazil known as "sempre-vivas" (everlasting flowers). In this work, we evaluated the potential anti-mycobacterial activity of two methoxylated flavonoids (flavonoid 7-methylquercetagetin and 7-methylquercetagetin-4'-O-β-D-glucopyranoside) isolated and identified from P. latipes and the naphthopyranone fractions from P. bromeliodes ethanolic extracts. The MIC value of 500 µg/mL was verified for all compounds tested against M. tuberculosis H37Rv. For M. avium, the MIC value ranged from 1000-2000 µg/mL excepting to naphthopyranone fractions with MIC of 500 µg/mL. This is the first report of activity determination of Paepalanthus spp. flavonoids activity against Mycobacterium tuberculosis and M. avium.

Thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide/hydrazones: anti-Mycobacterium tuberculosis activity and cytotoxicity.

The aim of this study was to identify a candidate drug for the development of anti-tuberculosis therapy from previously synthesized compounds based on the thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide/hydrazones compounds. The minimal inhibitory concentration (MIC) of these compounds against Mycobacterium tuberculosis was determined. Their in vitro cytotoxicity to J774 cells (IC50) was determined to establish a selectivity index (SI) (SI=IC50/MIC). The best compounds were the thiosemicarbazones (2, 3 and 4) and the hydrazide/hydrazones (14, 15, 16 and 18). The results are comparable to or better than those of "first line" or "second line" drugs commonly used to treat TB, suggesting these compounds as anti-TB drug candidates.

Ruthenium (II) phosphine/picolinate complexes as antimycobacterial agents.

The synthesis, characterization and the anti-Mycobacterium tuberculosis (MTB) activities of three ruthenium complexes containing the 2-pyridinecarboxylic acid anion (picolinate), with formulae cis- [Ru(pic)(dppm)(2)]PF(6) (1), cis- [Ru(pic)(dppe)(2)]PF(6) (2) and [Ru(pic)(2)(PPh(3))(2)] (3) [pic=2-pyridinecarboxylate; dppm=bis(diphenylphosphino)methane; dppe=1,2-bis(diphenylphosphino)ethane; PPh(3)=triphenylphosphine] are reported in this article. The complexes were characterized by elemental analysis, spectroscopic and electrochemical techniques. Their in vitro antimycobacterial activity was determinated as the Minimum Inhibitory Concentration (MIC) for MTB cell growth, measured by the REMA method. The best MICs were found for complexes (1) and (2), with values of 0.78 and 0.26 microg/mL, respectively. The results are comparable to or better than "first line" or "second line" drugs commonly used in the treatment of TB.

In vitro anti-Mycobacterium tuberculosis activity of some Brazilian "Cerrado" plants / Atividade anti-Mycobacterium tuberculosis in vitro de algumas plantas do "Cerrado" Brasileiro

O objetivo deste trabalho foi realizar uma seleção de algumas plantas de uma determinada região Brasileira com atividade contra Mycobacterium tuberculosis. Extratos clorofórmicos e metanólicos de 37 espécies de plantas distribuídas em 17 famílias do "Cerrado" Brasileiro foram avaliadas contra M. tuberculosis H37Rv e a Concentração Inibitória Mínima (CIM) foi determinada pelo uso do Microplate Alamar Blue Assay (MABA). Extratos brutos de dezesseis plantas apresentaram valor de CIM < 125 µg/mL e três de 31,2 µg/mL. Estes resultados sugerem que o "Cerrado" Brasileiro deve possuir um recurso de plantas com constituintes ativos anti-M. tuberculosis que podem ser extraídos por solventes polares e apolares.

The aim of this work was to carry out a screening of some plants of this Brazilian region with activity against Mycobacterium tuberculosis. Chloroform and methanol extracts of 37 plant species distributed among 17 families from Brazilian "Cerrado" which were tested against M. tuberculosis H37Rv and the Minimum Inhibitory Concentration (MIC) was determined by the use of Microplate Alamar Blue Assay (MABA). Crude extracts from sixteen plants showed MIC value of < 125 µg/mL and three 31.2 µg/mL. These results suggest that the Brazilian "Cerrado" may be a source of plants that have activity anti-M. tuberculosis constituents that can be extracted by polars and apolars solvents.

Antimycobacterial activity of natural and semi-synthetic lignans.

The antimycobacterial activity of (-)-cubebin (1), hinokinin (2), and some of their semisynthetic derivatives, namely (-)-O-acetyl-cubebin (3), (-)-O-methyl-cubebin (4), (-)-O-(N,N-dimethylamine-ethyl)-cubebin (5) and (-)-6,6'-dinitrohinokinin (6), was evaluated against Mycobacterium tuberculosis (ATCC 27294), M. kansasii (ATCC 12478), and M. avium (ATCC 15769). The MIC values ranged from 31.25 to 2000 microg/mL. Among the evaluated compounds, 2 displayed a MIC value of 62.5 microg/mL against M. tuberculosis, while 3 and 4 displayed MIC values of 62.5 and 31.25 microg/mL, respectively, against M. avium. All compounds were inactive against M. kansasii. These are promising results concerning the search for biologically active natural products, highlighting that new approaches to the prevention, treatment, and cure of tuberculosis are extremely important.

Synthesis, characterization, X-ray structure and in vitro antimycobacterial and antitumoral activities of Ru(II) phosphine/diimine complexes containing the "SpymMe2" ligand, SpymMe2=4,6-dimethyl-2-mercaptopyrimidine.

The reaction of cis-[RuCl(2)(dppb)(N-N)], dppb=1,4-bis(diphenylphosphino)butane, complexes with the ligand HSpymMe(2), 4,6-dimethyl-2-mercaptopyrimidine, yielded the cationic complexes [Ru(SpymMe(2))(dppb)(N-N)]PF(6), N-N=bipy (1) and Me-bipy (2), bipy=2,2'-bipyridine and Me-bipy=4,4'-dimethyl-2,2'-bipyridine, which were characterized by spectroscopic and electrochemical techniques and X-ray crystallography and elemental analysis. Additionally, preliminary in vitro tests for antimycobacterial activity against Mycobacterium tuberculosis H37Rv ATCC 27264 and antitumor activity against the MDA-MB-231 human breast tumor cell line were carried out on the new complexes and also on the precursors cis-[RuCl(2)(dppb)(N-N)], N-N=bipy (3) and Me-bipy (4) and the free ligands dppb, bipy, Me-bipy and SpymMe(2). The minimal inhibitory concentration (MIC) of compounds needed to kill 90% of mycobacterial cells and the IC(50) values for the antitumor activity were determined. Compounds 1-4 exhibited good in vitro activity against M. tuberculosis, with MIC values ranging between 0.78 and 6.25microg/mL, compared to the free ligands (MIC of 25 to >50microg/mL) and the drugs used to treat tuberculosis. Complexes 1 and 2 also showed promising antitumor activity, with IC(50) values of 0.46+/-0.02 and 0.43+/-0.08microM, respectively, against MDA-MB-231 breast tumor cells.

Synthesis, characterization and antimycobacterial activity of Ag(I)-aspartame, Ag(I)-saccharin and Ag(I)-cyclamate complexes.

The present work describes the synthesis and antimycobacterial activity of three Ag(I)-complexes with the sweeteners aspartame, saccharin, and cyclamate as ligands, with the aim of finding new candidate substances for fighting tuberculosis and other mycobacterial infections. The minimal inhibitory concentration of these three complexes was investigated in order to determine their in-vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium malmoense, and Mycobacterium kansasii. The MIC values were determined using the Microplate Alamar Blue Assay. The best MIC values found for the complexes were 9.75 microM for Ag(I)-aspartame against M. kansasii and 15.7 microM for Ag(I)-cyclamate against M. tuberculosis.

Synthesis, X-ray structure and antimycobacterial activity of silver complexes with alpha-hydroxycarboxylic acids.

In this paper, synthesis, characterization and antimycobacterial properties of a new water-soluble complex identified as silver-mandelate are described. Elemental and thermal analyses are consistent with the formula [Ag(C(6)H(5)C(OH)COO)](n). The polymeric structure was determined by single X-ray diffraction and the two-dimensional structure is based on the bis(carboxylate-O,O') dimer [Ag-O, 2.237(3), 2.222(3) Angstrom]. The structure is extended along both the b and c axes through two oxygen atoms of a bidentate alpha-hydroxyl-carboxylate residue [Ag-OH(hydroxyl), 2.477(3) Angstrom; Ag-O(carboxylate), 2.502(3) Angstrom; O-Ag-O, 63.94(9) degrees]. A strong d(10)-d(10) interaction was observed between two silver atoms. The Ag - Ag distance is 2.8307(15) Angstrom. The NMR (13)C spectrum in D(2)O shows that coordination of the ligand to Ag(I) occurs through the carboxylate group in solution. Potentiometric titration shows that only species with a molar metal:ligand ratio of 2:2 are formed in aqueous solution. The mandelate complex and the silver-glycolate, silver-malate and silver-hydrogen-tartarate complexes were tested against three types of mycobacteria, Mycobacterium avium, Mycobacterium tuberculosis and Mycobacterium kansasii, and their minimal inhibitory concentration (MIC) values were determined. The results show that the four complexes are potential candidates for antiseptic or disinfectant drugs for discharged secretions of patients affected with tuberculosis.

Potential tuberculostatic agent: micelle-forming pyrazinamide prodrug.

Pyrazinamide was condensed with the poly(ethylene glycol)-poly(aspartic acid) copolymer (PEG-PASP), a micelle-forming derivative was obtained that was characterized in terms of its critical micelle concentration (CMC) and micelle diameter. The CMC was found by observing the solubility of Sudan III in Poly(ethylene glycol)-poly(pyrazinamidomethyl aspartate) copolymer (PEG-PASP-PZA) solutions. The mean diameter of PEG-PASP-PZA micelles, obtained by analyzing the dynamic light-scattering data, was 78.2 nm. The PEG-PASP-PZA derivative, when assayed for anti-Mycobacterium activity, exhibited stronger activity than the simple drug.

Antioxidant and antimycobacterial activities of Tabernaemontana catharinensis extracts obtained by supercritical CO2 + cosolvent.

In the present work the antioxidant and antimycobacterial activities were determined for extracts from Tabernaemontana catharinensis. The extracts' global yields were obtained using supercritical CO2 plus cosolvent. The cosolvents ethanol, isopropyl alcohol, methanol, and water and their mixtures were used. The extracts were fractionated and analyzed by thin-layer chromatography and gas chromatography/flame ionization detection. The antimycobacterial activity was measured against Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium kansasii. The antioxidant activity was determined by the coupled reaction of beta-carotene and limonene acid. The average global yield was approximately constant (2.4 +/- 0.1%) for the alcoholic cosolvents and significantly larger (15 +/- 1%) for the cosolvent water and its alcoholic mixtures. The content of alkaloids in the extracts was strongly affected by the cosolvent. The antioxidant activity of the extracts ranged from 53% to 95%. The highest antimycobacterial activity was detected in the alkaloidal fraction (minimum inhibitory concentration = 128 microg/mL), while the lowest was verified in the aqueous fraction (minimum inhibitory concentration >512 microg/mL).

Neo-clerodane diterpenoid, a new metalloprotease snake venom inhibitor from Baccharis trimera (Asteraceae): anti-proteolytic and anti-hemorrhagic properties.

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. Few attempts have been made however to identify the nature of plant natural products with anti-ophidian properties. Baccharis trimera (Less) DC (Asteraceae), known in Brazil as carqueja, has been popularly used to treat liver diseases, rheumatism, diabetes, as well as digestive, hepatic and renal disorders. The active component was identified as 7alpha-hydroxy-3,13-clerodadiene-16,15:18,19-diolide, C20H28O5, (clerodane diterpenoid, Bt-CD). We report now the anti-proteolytic and anti-hemorrhagic properties against snake venoms of a Bt-CD inhibitor from B. trimera. Bt-CD exhibited full inhibition of hemorrhage and proteolytic activity caused by Bothrops snake venoms. The inhibitor was able to neutralize the hemorrhagic, fibrinogenolytic and caseinolytic activities of class P-I and III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. No inhibition of the coagulant activity was observed. Bt-CD also partially inhibited the edema induced by other crude venoms, metalloproteases, basic and acidic phospholipases A2. To further elucidate the inhibitory specificity of Bt-CD against metalloproteases isolated from snake venoms, a deeper understanding of its structure and function is necessary. Furthermore, the potential use of these inhibitors to complement anti-venom as an alternative treatment of snakebite envenomations needs to be evaluated in future studies.

Screening and characterization of mutations in isoniazid-resistant Mycobacterium tuberculosis isolates obtained in Brazil.

We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide -48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G(269)A substitution. Although KatG(315) substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.

Functional properties of spice extracts obtained via supercritical fluid extraction.

In the present study the antioxidant, anticancer, and antimycobacterial activities of extracts from ginger (Zingiber officinale Roscoe), rosemary (Rosmarinus officinalis L.), and turmeric (Curcuma longa L.) were evaluated. The extracts were obtained using supercritical CO(2) with and without ethanol and/or isopropyl alcohol as cosolvent. The extracts' antioxidant power was assessed using the reaction between beta-carotene and linolenic acid, the antimycobacterial activity against M. tuberculosis was measured by the MABA test, and their anticancer action was tested against nine human cancer ancestries: lung, breast, breast resistant, melanoma, colon, prostate, leukemia, and kidney. The rosemary extracts exhibited the strongest antioxidant and the lowest antimycobacterial activities. Turmeric extracts showed the greatest antimycobacterial activity. Ginger and turmeric extracts showed selective anticancer activities.

Isolamento de micobactérias em espécimes clínicos, utilizando o sistema automatizado MB/BacT-TM / Mycobacteria isolation in clinical specimens using the MB/BacT-TM system

O aumento da incidência da tuberculose e outras micobacterioses têm demonstrado a importância de se isolar e identificar rapidamente as micobactérias. No período de março a dezembro de 2000 foram avaliados 791 espécimes clínicos coletados de pacientes com sorologia positiva para HIV. O sistema MB/BacTTM detectou 30,0 por cento de amostras positivas, enquanto o meio Lowenstein Jensen 19,0 por cento. As identificações das micobactérias foram realizadas pelo sistema molecular de DNA AccuProbe ou por provas fenotípicas. O Sistema Automatizado MB/BacTTM foi mais sensível e rápido para o isolamento de micobactérias que o método tradicional e, acoplado a um sistema de identificação molecular, poderá ser uma ferramenta útil para o Programa de Controle da Tuberculose

3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N, N-dimethyl-3-(2-thienyl)-2-propen-1-amine: synthesis, cytotoxicity, and leishmanicidal, trypanocidal and antimycobacterial activities.

Current therapies for Chagas' disease, leishmaniasis and tuberculosis are unsatisfactory because of the failure rates, significant toxicity and/or drug resistance. In this study, the compound 3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N,N-dimethyl-3-(2-thienyl)-2-propen-1-amine (IV) was synthesized and its trypanocidal, leishmanicidal and antimycobacterial activities were investigated. The cytotoxicity was determined on V79 cells with three endpoints: nucleic acid content, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide reduction and Neutral Red uptake. This compound was active against different species of mycobacteria and different life cycle stages of Trypanosoma cruzi. In experiments with trypomastigotes performed at 4 degrees C in the presence of blood, the activity was 8.8-fold more active than the standard drug, Crystal Violet. Higher activity was achieved against Leishmania amazonensis, with an ED(50)/24 h of 3.0 +/- 0.3 micro mol/L. The effect against trypanosomatids, which suggests high activity of compound IV against promastigotes of L. amazonensis and amastigotes of T. cruzi, stimulated further studies in vitro with amastigotes interiorized in macrophages and with in vivo models. Our results indicate that mammalian V79 cells are less susceptible to the action of compound IV than promastigotes of L. amazonensis (8.0-13.3-fold) and axenic amastigotes of T. cruzi (3.5-5.9-fold).

Problemas na padronização da reação em cadeia da polimerase para diagnóstico da tuberculose pulmonar / Problems in the standardization of the polymerase chain reaction for the diagnosis of pulmonary tuberculosis

Objetivo - Padronizar em cadeia da polimerase para diagnóstico de tuberculose pulmonar, comparando os resultados obtidos com as técnicas microbiológicas clássicas, e analisar seu uso numa região de alta prevalência da tuberculose. Métodos - Foram descontaminadas, após a baciloscopia, 42 amostras de escarro de pacientes. Em seguida, procedeu-se ao cultivo em Lowenstein-Jensen e à reação em cadeia da polimerase com "primers" que amplificam um fragmento de 123 pares de base do genoma do Mycobacterium tuberculosis. Resultados - Das 42 amostras de escarro, 10 apresentaram cultura positiva para M. tuberculosis. Dez foram positivas à baciloscopia e 16 mostraram-se positivas na reação em cadeia da polimerase. A sensibilidade e especificidade do teste em relação à cultura foi de 90 por cento e 81 por cento, respectivamente. Conclusões - A reação em cadeia da polimerase tem sensibilidade comparável à da cultura e pode ser realizada em apenas um dia, resultado em tratamento precoce e melhor controle da doença. A padronização e avaliação de técnica de biologia molecular no diagnóstico da tuberculose no Brasil é imprescindível na discussão da implantação deste exame na rotina diagnóstica em centros de referência.