Isoniazid and verapamil modulatory activity and efflux pump gene expression in Mycobacterium tuberculosis.

INTRODUCTION: Resistance to first-line anti-tuberculosis drugs is a major concern in the treatment of the disease. New strategies, such as the use of efflux pump inhibitors (EPIs), are being investigated to improve the outcome of the treatment. Verapamil (VP), one such inhibitor, was shown to inhibit several efflux pump (EP) Mycobacterium tuberculosis proteins and demonstrate synergic activity with anti-TB drugs.OBJECTIVE: To evaluate the combinatory effect of isoniazid (INH) and VP in M. tuberculosis.METHODS: Minimal inhibitory concentrations and combinatory effects of INH+VP were determined using respectively resazurin microtitre assay plate (REMA) and resazurin drugs combination microtitre assay (REDCA). From the results, we selected three bacilli with different susceptibility profiles and assessed their expression of 10 EP genes through quantitative reverse transcription polymerase chain reaction after exposure to INH, VP and INH + VP for 48 h.RESULTS: A significant reduction of INH MIC was observed in INH-susceptible isolates upon combination with VP. In brief, gene expression assays revealed expression patterns that could be correlated with each resistance profile, presence or absence of gene mutations and combinatory effect with VP.CONCLUSION: Combining VP with INH showed important results in drug-susceptible strains, and clinical trials on combined VP + anti-TB drugs should be discussed.

Effects of treatments for drooling on caries risk in children and adolescents with cerebral palsy.

BACKGROUND: Neuromuscular impairment makes individuals with cerebral palsy (CP) more prone to drooling. Among the treatment options, there are procedures that interfere with saliva production. It is imperative to evaluate the effect of the different modalities since the reduction in salivary flow rate/production may exacerbate the risk of dental caries. MATERIAL AND METHODS: The aim of this study was to compare the effects of different treatments for drooling on caries risk and salivary parameters in children and adolescents with CP. STUDY DESIGN: A total of 142 children and adolescents with CP, aged 6 to 18 years, were assigned to groups based on the different treatments they had received for drooling: G1-anticholinergic drugs (n = 18), G2-botulinum toxin injection (n = 16), G3-salivary glands surgery (n = 16), G4-no treatment (n = 42), and G5-non-drooling subjects (n = 50). All participants were evaluated on the Simplified Oral Hygiene Index, and for the prevalence of dental caries (decayed, missing, and filled teeth index and white spot lesions). Unstimulated whole saliva was collected, and salivary flow rate and osmolality were measured. Chi-square, ANOVA and Poisson regression were calculated. Prevalence ratios and their respective 95 % confidence intervals were obtained. The significance level was fixed at 5%. RESULTS: No differences were found in the decayed, missing, and filled teeth index (p = 0.128) and Simplified Oral Hygiene Index (p = 0.674) among the different groups. G3 presented significantly higher percentages of WSL (p < 0.001), lower values of salivary flow rate (p < 0.001), and higher values of osmolality (p < 0.001). The white spot lesion prevalence ratio was higher only for G3 (Prevalence ratio = 14.36; IC 95% = 4.64-44.40; p < 0.001). CONCLUSIONS: Children and adolescents with CP who had received surgical treatment for drooling exhibited higher number of white spot lesions because of the reduced salivary flow rate and higher salivary osmolality.

Pyrazinamide susceptibility testing in Mycobacterium tuberculosis using the fast resazurin microtiter assay plate.

SETTING: Department of Clinical Analysis and Biomedicine, State University of Maringa, Maringa, PR, Brazil. OBJECTIVE: To evaluate the performance of the resazurin microtiter assay (REMA) plate at pH 5.5 in detecting Mycobacterium tuberculosis susceptibility to pyrazinamide (PZA). DESIGN: The minimal inhibitory concentration (MIC) of PZA in M. tuberculosis H37Rv and M. bovis AN5 reference strains and in 34 clinical M. tuberculosis isolates (26 PZA-susceptible and eight PZA-resistant) was determined using REMA at pH 5.5 and compared to REMA at pH 6.0. RESULTS: REMA at pH 5.5 was helpful in discriminating PZA-susceptible from resistant M. tuberculosis isolates when â©¿50 µg/ml PZA was considered as the cut-off for PZA susceptibility. Furthermore, it provided results in 8 days. However, two PZA-resistant isolates failed to grow at pH 5.5. CONCLUSION: As the REMA method is rapid, inexpensive, easy to perform and read, it would be of great usefulness in low-income countries for detecting PZA-resistant M. tuberculosis. REMA at pH 5.6-5.9 should be evaluated on an extended panel of clinical M. tuberculosis isolates with a greater range of MIC values in different laboratories for a better understanding of its utility in differentiating PZA-resistant from PZA-susceptible isolates.

Potential of Casiopeínas® Copper Complexes and Antituberculosis Drug Combination against Mycobacterium tuberculosis.

New compounds with antituberculosis activity and their combination with classic drugs have been evaluated to determine possible interactions and antagonism. The aim of this study was to evaluate the in vitro activity of Casiopeínas® copper-based compounds (CasIIIia, CasIIIEa, and CasIIgly) alone and combined with isoniazid (INH), rifampicin, or ethambutol (EMB) against resistant and susceptible Mycobacterium tuberculosis. Seventeen clinical M. tuberculosis isolates (5 multi-drug resistant and 2 resistant to INH and/or EMB) were subjected to determination of the minimal inhibitory concentration (MIC) by the resazurin microtiter assay and combination assessment by the resazurin drug combination microtiter assay. The Casiopeínas® alone showed a remarkable effect against resistant isolates with MIC values from 0.78 to 12.50 µg/ml. Furthermore, a synergistic effect mainly with EMB is shown for both resistant and susceptible clinical isolates. Casiopeínas® are promising candidates for future investigation into the development of antituberculosis drugs, being one of the first examples of essential metal-based drugs used in this field.

Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex.

We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

In vitro interaction of eupomatenoid-5 from Piper solmsianum C. DC. var. solmsianum and anti-tuberculosis drugs.

SETTING: Department of Clinical Analysis and Biomedicine, State University of Maringa, Maringa, Parana, Brazil. OBJECTIVE: To evaluate the in vitro interaction between eupomatenoid-5 (EUP-5), extracted from Piper solmsianum C. DC. var. solmsianum, and first-line anti-tuberculosis drugs against Mycobacterium tuberculosis H37Rv and 20 clinical isolates. DESIGN: Resazurin drugs combination microtiter assay (REDCA) was performed to determine the interaction between EUP-5 and isoniazid, rifampicin (RMP) and ethambutol (EMB). RESULTS: Synergism was observed in M. tuberculosis H37Rv and eight clinical isolates with EUP-5+RMP, and in M. tuberculosis H37Rv and 17 clinical isolates with EUP-5+EMB combinations. CONCLUSION: EUP-5 is a promising compound for further studies on the development of anti-tuberculosis drugs.

Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex

We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

Anti-tuberculosis neolignans from Piper regnellii.

The present study determined the anti-Mycobacterium tuberculosis activities of supercritical CO2 extracts, neolignans eupomatenoid-5 (1), conocarpan (4) and eupomatenoid-3 (7) and their derivatives (2, 3, 5, 6, and 8) from Piper regnellii, as well as their cytotoxicities. The supercritical CO2 extract from leaves was purified by chromatographic methods, yielding compounds (1), (4) and (7), which were identified by (1)H NMR and comparison with literature data. Anti-M. tuberculosis activity (H37Rv and clinical isolates) was evaluated using a resazurin microtiter assay plate (REMA) to determine the MIC. The cytotoxicity assay was carried out in macrophages J774G.8 by sulforhodamine B colorimetric assay. The supercritical CO2 extracts from leaves and stems, and compound (4) showed activity against M. tuberculosis (MIC 15.6 µg/ml). Compound (1) showed the best activity (MIC 1.9 µg/ml), with good SI. Compounds (7) and (8) showed low activity against M. tuberculosis H37Rv. The derivative compounds did not show increased anti-M. tuberculosis activity. This is the first report, to our knowledge, to describe neolignans from P. regnellii with activity against M. tuberculosis, and compound (1) is a potential candidate for future antituberculosis drugs.

Use of the polymerase chain reaction to detect Mycobacterium leprae in urine

Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients’ urine samples was successfully amplified by PCR-Pra in 46.6 percent (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75 percent for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30 percent for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.

Use of the polymerase chain reaction to detect Mycobacterium leprae in urine.

Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients' urine samples was successfully amplified by PCR-Pra in 46.6% (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75% for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30% for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.

Occurrence and antibiotic resistance of coliform bacteria and antimicrobial residues in pasteurized cow's milk from Brazil.

In this study we examined the hygienic and sanitary quality of pasteurized cow's milk in the state of Paraná, Brazil, by determining the presence of coliforms and occurrence of antimicrobial residues. A total of 260 milk samples were collected from commercial establishments in different regions of the state. Coliform populations were estimated by the multiple-tube test, and antimicrobial residues were detected by enzyme-linked immunosorbent assay. Overall, 105 samples (40.4%) were unsuitable for consumption according to Brazilian legal standards. Among the coliforms, Escherichia coli and Klebsiella pneumoniae were respectively identified in 77.05 and 36.07% of the samples. The highest rates of resistance to antimicrobial agents were observed for ampicillin (19.2%), cephalothin (18.9%), and tetracycline (17.1%). Antimicrobial residues were detected in 80 samples (30.8%). Forty-eight samples (18.5%) were positive for tetracycline, 29 (17.4%) for neomycin, 9 (3.5%) for beta-lactams, 6 (2.3%) for gentamicin, 4 (1.5%) for chloramphenicol, and 1 (0.4%) for streptomycin-dihydrostreptomycin. The results demonstrate a high prevalence of coliforms and also a high occurrence of antimicrobial residues in pasteurized cow's milk from Paraná, Brazil.

Direct detection of Mycobacterium bovis in bovine lymph nodes by PCR.

Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff's method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 microl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Comparative effect of physiological levels of leptin and insulin on cyclic AMP-induced stimulation of hepatic glycogen breakdown.

Physiological level of insulin showed more prominent inhibiting effect on the activation of hepatic glucose production and glycogenolysis promoted by cAMP than physiological levels of leptin.

Simplified technique for detection of significant bacteriuria by microscopic examination of urine.

A comparative study of microscopic examination of 10 microl (simplified loop technique) and 50 microl (traditional drop technique) of uncentrifuged Gram-stained urine specimens for detection of significant bacteriuria was carried out. The results demonstrated that the 10-microl loop technique can be used as an alternative to the 50-microl drop technique for presumptive diagnosis of urinary-tract infection in bacteriological practice, with the advantages of greater rapidity and ease of performance.

[Frequency of anti-Cysticercus cellulosae antibodies in individuals from five counties in the Northern region of Brazil]. / Freqüência de anticorpos anti-Cysticercus cellulosae em indivíduos de cinco municípios da região Norte do Estado do Paraná-Brasil.

An epidemiological and serological study was carried out on a sample of 2,180 individuals, in five counties in the north of Paraná State-Brazil, using the indirect immunofluorescence test to detect anti-Cysticercus cellulosae antibodies. These individuals, 69 (3.2%) showed significant titers of antibodies. No single significant difference between the proportion of reactivity in Sarandi (6.6%) and in Marialva (4.7%) was observed (Z = 1,319, P = 0.0936), but it was significantly higher than that observed in Mandaguaçu, Paiçandu and Maringá (P < 0.01). Of these individuals, 47.9% were within 21-49 years old and 79.4% were of female sex. "Headache" (70.6%), "faintness" (57.4%), and "convulsions" (7.4%) were among the most frequent by reported, moreover, cases of Taenia infections (22.1%) and the custom of eating uncooked beef (41.2%) or pork (27.9%) and meat containing cysticerci (25.0%) were also related.

A medium for inducing conversion of Histoplasma capsulatum var. capsulatum into its yeast-like form

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of wich were conventional and three novel. One of our novel media, MLGema, induced complete conversion, of two strains within five days of incubation at 35 degrees centigrades, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce

A medium for inducing conversion of Histoplasma capsulatum var. capsulatum into its yeast-like form.

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of which were conventional and three novel. One of our novel media, MLGema, induced complete conversion of two strains within five days of incubation at 35 degrees C, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce.