Rapid Molecular Detection of Tuberculosis and Rifampicine Resistance in Ecuador

Background: In Ecuador, tuberculosis (TB) remains a serious problem that is complicated by the emergence of multidrug-resistant TB (MDR-TB). To evaluate this problem, this study was carried out at the Social Security Hospital (IESS) in Guayaquil, Ecuador from 2013 to 2015. Methods: The Xpert TB/RIF system was used to detect TB and MDR-TB and a survey was carried out to identify the factors that are potentially causing MDR-TB. Findings: 200 TB patients were confirmed on 5649 suspected patients and 20 (10%) with MDR-TB. It was observed that the annual prevalence of TB and MDR-TB had declining during study period. Trends have been declining but co-infection has doubled since 2009 with 16% of patients co-infected with HIV. Potential resistance factors identified were: disruption in drug supply, lack of resources and lack of credibility of treatment.

Feasibility of Integrating Leprosy Post-Exposure Prophylaxis with Single-Dose Rifampicin (LPEP) into Routine Leprosy Control Program in Bukedi and Teso Regions in Uganda

Background: World Health Organization recommends the implementation of contact tracing and Leprosy Post Exposure prophylaxis (LPEP) to interrupt the chain of transmission. To accelerate the uptake of this recommendation, a cross-sectional study among contacts of leprosy patients was conducted to investigate the feasibility of integrating leprosy systematic contact tracing and post-exposure prophylaxis (PEP) into the routine leprosy control program. Methods: This was a mixed methods cross-sectional study. The study was implemented in Kumi, Ngora, Serere, Soroti, Budaka and Kibuku Districts. Results: The 45 enrolled index patients (97.8% of the registered) identified a total of 135 contacts, of which 134 (99·2%) consented and were screened. Among them, one new leprosy patient was identified and started on treatment with multidrug therapy (MDT). All the eligible contacts, received the prophylactic treatment with Single Dose Rifampicin (SDR). Overall, SDR was administered to 133(98.5% of the listed contacts) with no adverse event reported. Factors associated with successful contact investigation and management included: Involvement of index patients, health care workers during the contact screening and SDR A administration, counselling of the index patients and contacts by the health care works, LPEP being administered as Directly observed Therapy (DOT) among others. Results Interpretation: The integration of leprosy post-exposure prophylaxis with administration of SDR and contact tracing is feasible, generally accepted by the patient, their contacts and health workers and can be integrated into the National Leprosy control programmes with minimal additional efforts once contact tracing has been established. Therefore, we recommend integration of administration of SDR in to the routine leprosy control program.

Resistant Pulmonary TB-HIV Co-Infection in an Infant: About a Case

Diagnosis of childhood tuberculosis (TB) is difficult, especially in resource-limited countries where the number of reported cases of TB-HIV co-infection continues to rise. This co-infection poses a diagnostic and therapeutic problem for caregivers. We report a case of rifampicin-resistant HIV-TB pulmonary coinfection in a 19-month-old infant.

Tuberculosis in kidney transplant candidates and recipients

Tuberculosis(TB)is the leading cause of infectious mortality and morbidity in the world,second only to coronavirus disease 2019.Patients with chronic kidney disease and kidney transplant recipients are at a higher risk of developing TB than the general population.Active TB is difficult to diagnose in this population due to close mimics.All transplant candidates should be screened for latent TB infection and given TB prophylaxis.Patients who develop active TB pre-or post-trans-plantation should receive multidrug combination therapy of antitubercular therapy for the recommended duration with optimal dose modification as per glomerular filtration rate.

The Contribution of the Xpert® MTB/RIF Assay to the Surveillance of Drug-Resistant Tuberculosis in the Central African Republic

Introduction: The Central African Republic is one of the 30 high Tuberculosis burden countries in the world, with an incidence of 540 cases per 100,000 population and a mortality of 91 deaths per 100,000 population. Since 2020, following WHO recommendations, the National Reference Laboratory for Tuberculosis has been using the Xpert® MTB/RIF assay as a first-line diagnostic test for the early detection of Drug Resistance Tuberculosis. The goal of this study was to evaluate the contribution of the Xpert® MTB/RIF assay to the surveillance of rifampicin resistance in new and previously treated tuberculosis cases. Materials and Methods: The data relative to the Xpert® MTB/RIF assay carried out on various categories of tuberculosis patients registered at the National Reference Laboratory for Tuberculosis in 2020 were analyzed retrospectively. The categories of tuberculosis patients were new cases, failed treatment cases, relapse cases, lost-to-follow-up cases and multidrug-resistant tuberculosis contact cases. Results: A total of 1404 tuberculosis patients were registered at the NRL-TB in 2020;the mean age was 39.2 years (2 - 90 years) and the male-to-female sex ratio was 1.16:1. Overall, 32.7% (454/1404) proved infected with tuberculosis, of which 22.5% (102/454) cases showed resistance to rifampicin. The primary resistance rate was 9.1% (27/298) and the secondary resistance rate was 46.6% (75/161). Treatment failures and relapsed cases were significantly associated with rifampicin resistance (p 0.005). Conclusion: Large-scale use of Xpert® MTB/RIF, especially in the provinces of the Central African Republic, will help the Ministry of Health to better control Drug Resistance Tuberculosis in the country.

Development and Validation of Liquid Chromatographic Method for the Determination of the Related Substances of Rifampicin

A number of HPLC methods have been approved for the determination of rifampicin, but no references involved the assay of related substances of rifampicin. In the present paper, a reversed-phase liquid chromatographic method for the determination of related substances of rifampicin is developed and validated. Rifampicin and its related substances, including rifamycin SV, rifampicin N-oxide and 3-formylrifamycin SV were separated using a Zorbax Eclipse C8, 250×4.6 mm(i.d), 5 μm column by isocratic elution at a flow rate of 1 mL·min -1. The detector was set at 254 nm. The mobile phase is a mixture of methanol-acetonitrile -0.075 mol·L -1 monopotassium phosphate -1.0 mol·L -1 citric acid (31:31:35:3, v/v). The method validation included accuracy, precision, linearity, sensitivity and stability. All results are shown to be acceptable.

Effect of garlic on isoniazid and rifampicin-induced hepatic injury in rats

AIM： To evaluate the hepatoprotective effect of garlic on liver injury induced by isoniazid （INH） and rifarnpicin （RIF）. METHODS： Wistar rats weighing 150-200 g were treated orally with 50 mg/kg of INH and RIF daily each for 28 d. For hepatoprotective studies, 0.25 g/kg per day of freshly prepared garlic hornogenate was administered orally half an hour before the INH＋RIF doses. Serum alanine arninotransferase （AIT）, aspartate aminotransferase （AST） and bilirubin were estimated on d 0, 14, 21, and 28 in all the rats. Histological analysis was carried out to assess the injury to the liver. Lipid peroxidation （1PO） as a marker of oxidative stress and non-protein thiols （glutathione） for antioxidant levels were measured in liver hornogenate. RESULTS： The treatment of rats with INH＋RIF （50 mg/kg per day each） induced hepatotoxicity in all the treated animals as judged by elevated serum ALT, AST, and bilirubin levels, presence of focal hepatocytic necrosis （6/8） and portal triaditis （8/8）. Garlic simultaneously administered at a dose of 0.25 g/kg per day prevented the induction of histopathological injuries in INH＋RIF co-treated animals, except in 4 animals, which showed only moderate portal triaditis. The histological changes correlated with oxidative stress in INH＋RIF treated animals. The group which received 0.25 g/kg per day garlic hornogenate along with INH＋RIF showed higher levels of glutathione （P〈0.05） and low levels of 1PO （P〈 0.05） as compared to INH＋RIF treated group. CONCLUSION： Freshly prepared garlic homogenate protects against INH＋RIF-induced liver injury in experimental animal model.

Berberine prevents stress-induced gut inflammation and visceral hypersensitivity and reduces intestinal motility in rats

BACKGROUND Irritable bowel syndrome (IBS) is a common chronic non-organic disease of the digestive system. Berberine (BBR) has been used to treat patients with IBS, but the underlying therapeutic mechanism is little understood. We believe that BBR achieves its therapeutic effect on IBS by preventing stress intestinal inflammation and visceral hypersensitivity and reducing bowel motility. AIM To test the hypothesis that BBR achieves its therapeutic effect on IBS by preventing subclinical inflammation of the intestinal mucosa and reducing visceral hypersensitivity and intestinal motility. METHODS IBS was induced in rats via water avoidance stress (WAS). qRT-PCR and histological analyses were used to evaluate the levels of cytokines and mucosal inflammation, respectively. Modified ELISA and qRT-PCR were used to evaluate the nuclear factor kappa-B (NF-κB) signal transduction pathway. Colorectal distention test, gastrointestinal transit measurement, Western blot, and qRT-PCR were used to analyze visceral sensitivity, intestinal motility, the expression of Ckit (marker of Cajal mesenchymal cells), and the expression of brain derived neurotrophic factor (BDNF) and its receptor TrkB.RESULTS WAS led to mucosal inflammation, visceral hyperalgesia, and high intestinal motility. Oral administration of BBR inhibited the NF-κB signal transduction pathway, reduced the expression of pro-inflammatory cytokines [interleukin (IL)- 1β, IL-6, interferon-γ, and tumor necrosis factor-α], promoted the expression of anti-inflammatory cytokines (IL-10 and transforming growth factor-β), and improved the terminal ileum tissue inflammation. BBR inhibited the expression of BDNF, TrkB, and C-kit in IBS rats, leading to the reduction of intestinal motility and visceral hypersensitivity. The therapeutic effect of BBR at a high dose (100 mg/kg) was superior to than that of the low-dose (25 mg/kg) group. CONCLUSION BBR reduces intestinal mucosal inflammation by inhibiting the intestinal NF-κB signal pathway in the IBS rats. BBR reduces the expression of BDNF, its receptor TrkB, and C-kit. BBR also reduces intestinal motility and visceral sensitivity to achieve its therapeutic effect on IBS.

Naringenin protects against isoniazid- and rifampicininduced apoptosis in hepatic injury

AIM To explore the protective effects and mechanisms of naringenin(NRG) on hepatic injury induced by isoniazid(INH) and rifampicin(RIF).METHODS Male mice were randomly divided into four groups and treated for 14 d as follows: normal control group was administered intragastrically with normal saline solution alone; model group was administered intragastrically with INH(100 mg/kg) and RIF(100 mg/kg); lowand high-dosage NRG pretreatment groups were administered intragastrically with different doses of NRG(50 or 100 mg/kg) 2 h before INH and RIF challenge. Mice were killed 16 h after the last dose of drug treatment to determine activity of serum transaminases. Oxidative stress was evaluated by measuring hepatic glutathione(GSH) and superoxide dismutase(SOD) and malondialdehyde(MDA) levels. Histopathological changes in hepatic tissue were observed under the optical microscope. Hepatocyte apoptosis was measured by TUNEL assay and caspase-3 activation. Expression of Bcl-2 and Bax in liver was determined by western blot.RESULTS Both low- and high-dosage NRG pretreatment obviously alleviated serum levels of alanine aminotransferase and aspartate aminotransferase, liver index, hepatic MDA content, and increased hepatic GSH content and SOD activity compared with the INH and RIF-treated group(44.71 ± 8.15 U/L, 38.22 ± 6.64 U/L vs 58.15 ± 10.54 U/L; 98.36 ± 14.78 U/L, 92.41 ± 13.59 U/L vs 133.05 ± 19.36 U/L; 5.34% ± 0.26%, 4.93% ± 0.25% vs 5.71% ± 0.28%; 2.76 ± 0.67 nmol/mgprot, 2.64 ± 0.64 nmol/mgprot vs 4.49 ± 1.12 nmol/mgprot; 5.91 ± 1.31 mg/gprot, 6.42 ± 1.42 mg/gprot vs 3.11 ± 0.73 mg/gprot; 137.31 ± 24.62 U/mgprot, 148.83 ± 26.75 U/mgprot vs 102.34 ± 19.22 U/mgprot; all P < 0.01 or 0.05). Histopathological evaluation showed obvious necrosis and inflammatory cell infiltration in liver of mice administered INH and RIF; however, mice pretreated with NRG showed minor hepatic injury. In addition, INH and RIF resulted in hepatocyte apoptosis, and NRG pretreatment dramatically suppressed INHand RIF-induced hepatocytes apoptosis. Furthermore, NRG-mediated anti-apoptotic effects seemed to be in connection with its regulation of Bax and Bcl-2 protein expression in hepatic tissue.CONCLUSION NRG might attenuate INH- and RIF-induced hepatic injury via suppression of oxidative stress and hepatocyte apoptosis.

Hepatoprotective potential of ethanolic extract of Ziziphus oenoplia(L.)Mill roots against antitubercular drugs induced hepatotoxicity in experimental models

Objective:To evaluate the hepatoprotective potential of ethanolic(50%) extract of Ziziphus oenoplia(L.) Mill(Z.oenoplia) root against isoniazid(INH) and rifampicin(RIF) induced liver damage in animal models.Methods:Five groups of six rats each were selected for the study.Ethanolic extract at a dose of 150 and 300 mg/kg as well as silymarin(100 mg/kg) were administered orally once daily for 21 d in INH + RIF treated groups.The serum levels of glutamic oxaloacetic transaminase(SGOT),glutamate pyruvate transaminase(SGPT),alkaline phosphatase (SALP),and bilirubin were estimated along with activities of superoxide dismutase,catalase, glutathione S-transferase,glutathione peroxidase,and hepatic melondialdehyde formation. Histopathological analysis was carried out to assess injury to the liver.Result:The considerably elevated serum enzymatic activities of glutamic oxaloacetic transaminase,glutamate pyruvate transaminase,alkaline phosphatase and bilirubin due to INH + RIF treatment were restored towards norma) in a dose dependent manner after the treatment with ethanolic extract of Z.oenoplia roots.Meanwhile,the decreased activities of superoxide dismutase,catalase, glutathione S-transferase and glutathione peroxidase were also restored towards normal dose dependency.In addition,ethanolic extract also significantly prevented the elevation of hepatic melondialdehyde formation in the liver of INH + RIF intoxicated rats in a dose dependent manner. The biochemical observations were supplemented with histopathological examination of rat liver sections.Conclusions:The results of this study slrongly indicate that ethanolic extract of Z.oenoplia has a potent hepatoprotective action against INH + RIF induced hepatic damage in rats.

Luciferase reporter phage phAE85 for rapid detection of rifampicin resistance in clinical isolates of Mycobacterium tuberculosis

Objective:To evaluate luciferase reporter phage(LRP)phAE85 in rapid detection of rifampicin resistance in a region where TB is endemic.Methods:One hundred and ninety primary isolates on Lowenstein-Jensen medium were tested.Middlebrook 7H9 complete medium with and without rifampicin at 2μg/mL was inoculated with standard inoculum from suspensions of the clinical isolate.After incubation for 72 h,LRP was added.Following 4 h of further incubation,light output from both control and test was measured as relative light units.Strains exhibiting a reduction of less than 50%relative light units in the drug containing vial compared to control were classified as resistant.Results were compared with the conventional minimum inhibitory concentration method(MIC)of drug susceptibility testing.Results:The two methods showed high level of agreement of 97%(CI 0.94,0.99)and P value was 0.000 1.The sensitivity and specificity of LRP assay for detection of rifampicin resistance were 91%h(CI 0.75,0.98)and 99ct(CI0.95,1.00)respectively.Time to detection of resistance by LRP assay was 3 d in comparison with 28 d by the minimum inhibitory concentration method.Conclusions:LRP assay with phAE85 is 99%specific,91%sensitive and is highly reproducible.Thus the assay offers a simple procedure for drug sensitivity testing,within die scope of semi-automation.

Determination of Endophytic Bacteria Resistance against Rifampin and UV-mutagenesis

The study aimed to isolate and screen endophytic bacteria from the plant in order to understand their colonization dynamics in plants. [Method] 5 kinds of endophytic bacteda including H1, DP1, CJL1, DJL12 and YC1 were selected as the original strains, and conducted UV mutagenesis studies on H1 and DJL12 with weak resistance, while the resistance of them against rifampicin was confirmed. [Result] Through preliminary screening, five kinds of endophytic bacteria could grew well in the plate without rifampin, among them, H1 and DLJ12 were unable to survive under 10 μg/ml concentration of rifampicin, and the other three strains could survive under 50 μg/ml concentration of rifampicin. After UV mutagenesis, DLJ12 strain with dfampicin resistance concentration of 80 μg/ml was obtained. [ Conclusion] The study could provide theoretical and practical basis for development and utilization of endophytic bacteria.

Effect of rifampicin pre-and post-treatment on rotenone-induced dopaminergic neuronal apoptosis and alpha-synuclein expression

BACKGROUND： Rifampicin inhibits the formation of a-synuclein multimer and protects against 1-methyl-4-phenyl-1,2, 3, 6-tetrahydropyritine （MPTP）-induced PC12 cell apoptosis. OBJECTIVE： To compare the effect of rifampicin pre- and post-treatment on tyrosine hydroxylase and α-synuclein expression in substantia nigra pars compacta in a rat model of Parkinson＇s disease. DESIGN, TIME AND SE＇B＇ING： A randomized, controlled experiment was performed at the Experimental Animal Center of Sun Yat-sen University North Campus （China） from November 2006 to October 2008. MATERIALS： Rifampicin was purchased from MD, USA; rotenone was purchased from Sigma, USA; mouse anti-rat α-synuclein monoclonal antibody was purchased from B＆D, USA; and rabbit anti-rat tyrosine hydroxylase monoclonal antibody was purchased from Chemicon, USA. METHODS： A total of 72 male, Sprague Dawley rats, aged 8 weeks, were randomly assigned to 5 groups： blank control （n = 12）, rifampicin （n = 12）, rotenone （n = 16）, rifampicin pre-treatment （n = 16）, and rifampicin post-treatment （n = 16）. Parkinson＇s disease model rats were established via a subcutaneous injection of rotenone （1.5 mg/kg per day） in the three treatment groups, once a day for 3 successive weeks. Rifampicin （30 mg/kg per day） was intragastrically administered in the rifampicin pre-treatment group 3 days prior to rotenone induction and in the rifampicin post-treatment group 7 days after rotenone induction. Rats were treated with a subcutaneous injection of 1 mL/kg per day sunflower oil in the blank control group and an intragastric injection of 30 mg/kg per day rifampicin in the rifampicin group, once a day for 3 successive weeks in total. MAIN OUTCOME MEASURES： Prior to treatment and in the end of the 3^rd week after treatment, the rats were evaluated using the modified neurological severity score. The substantia nigra from the rats was extracted for hematoxylin-eosin staining. Western blot analysis was performed to determine tyrosine hydroxylase and α-synuclein expression. RESULTS： Hematoxylin-eosin staining revealed a significant reduction in the number of substantia nigral neurons in the rotenone group, in addition to neurodegradation, hypopigmentation, and pyknosis. In the rifampicin pre-treatment and post-treatment groups, the number of dopaminergic neurons was significantly increased compared with the rotenone group （P 〈 0.01）, with slight neuronal damage. Compared with the rotenone group, substantia nigral tyrosine hydroxylase expression was significantly increased in the rifampicin pre-treatment and post-treatment groups （P 〈 0.01）, but α-synuclein expression and modified neurological severity scores were significantly decreased （P 〈 0.01）. In addition, the effect of rifampicin in the pre-treatment group was superior to the post-treatment group. There was no significant difference in tyrosine hydroxylase and α-synuclein expression, or in the modified neurological severity scores, between the blank control and rifampicin groups （P 〉 0.05）. CONCLUSION： Rifampicin significantly attenuated neuropathological and behavioral motor deficits induced by rotenone. Moreover, rifampicin enhanced tyrosine hydroxylase expression, but inhibited α-synuclein expression. The effect of rifampicin pre-treatment was superior to rifampicin post-treatment.

Low Doses of Rifampicin Used in New Tuberculosis Patients Correlated to Increased Frequency of Rifampicin-Resistance and Poorer Treatment Outcomes

The prognosis of patients with previously treated tuberculosis (TB) was suggested to be dependent on whether the initial treatment was in compliance with the established guidelines. The aim of this retrospective multicenter study was to determine the proportion of new TB patients who received standard doses of rifampicin in multiple provinces of China, and the relationship between low doses of rifampicin and frequency of rifampicin-resistance as well as treatment outcomes. A total of 713 new TB patients were treated with either once-daily dose of bulk anti-TB drugs (group I) or every other day combination blister packs of anti-TB drugs containing rifampicin (group II) at more than 30 TB treatment centers/hospitals in China. Treatment history, therapeutic doses of rifampicin, and information about patients were extracted from their medical records and analyzed, and rifampicin-resistance of isolates collected from patients following the treatment as well as treatment outcomes were compared between two treatment groups. Among 522 patients in treatment group I, 154 (29.5%) received standard and 363 (69.5%) received low doses of rifampicin;238 (45.6%) isolates were rifampicin-resistant, and 243 (46.6%) were successfully treated. Among 191 patients in treatment group II, 175 (91.6%) received standard and 15 (7.9%) received low doses of rifampicin;72 (37.7%) isolates were rifampicin-resistant, and 105 (55%) were successfully treated. When patients who received low doses of rifampicin were compared to others within the same treatment group, increased rates for rifampicin-resistance and treatment failure were observed. Results from this study showed that most new TB patients in treatment group I (69.5%) received low doses of rifampicin, and their treatment outcomes were worse than those in treatment group II, indicating that low doses of rifampicin used for the initial treatment of new TB patients were correlated to increased frequency of rifampicin-resistance and poorer treatment outcomes.

Role of N-acetylcysteine in rifampicin-induced hepatic injury of young rats

AIM, To study the role of N-acetylcysteine （NAC） as a protective agent in rifampicin （RMP）-induced oxidative hepatic injury of young rats. METHODS： Hepatic injury was produced by giving 50mg/kg body weight/day of RMP for 3 wk. A dose of NAC （100mg/kg body weight/day） was given in combination with RMP intraperitoneally. Analysis of lipid peroxidation, thiol levels, cytochrome P4se, superoxide dismutase （SOD）, catalase, glutathione peroxidase, reductase and transferase were estimated in liver along with the body weight, liver weight and histological observations. RESULTS： RMP exposure resulted in no change in body and liver weight while antioxidative enzymes were altered but the non protein thiol （GSH） status was well preserved. Cytochrome P450 system and peroxidation of lipids were induced by RMP exposure. Partial protection was observed with NAC against RMP-induced changes in liver, which was evidenced from the prevention of increase in lipid peroxidation and the reduction in SOD and catalase enzyme levels. CONCLUSION. NAC protects young rats against RMP- induced oxidative hepatic injury.

Evaluation of antihepatotoxic potential of Solanum xanthocarpum fruit extract against antitubercular drugs induced hepatopathy in experimental rodents

Objective:To assess the hepatoprotective effect of Solanum xanthocarpum(S. xanthocarpum) fruit extract against antitubercular drug-induced liver toxicity in experimental animals.Methods:Ethanolic(50%) fruit extract ofS. xanthocarpum(100, 200 and 400 mg/kg bw) was administered daily for 35 days in experimental animals. Liver toxicity was induced by combination of three antitubercular drugs [isoniazid(I) 7.5 mg/kg, rifampicin(R) 10 mg/kg and pyrazinamide(P) 35 mg/kg] given orally as suspension for 35 days in rats. The hepatoprotective activity was assessed using various biochemical parameters like aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatise(ALP), total bilirubin(TBL), albumin(ALB), total protein(TP), lactate dehydroginase(LDH), and serum cholesterol(CHL). Meanwhile,in vivoantioxidant activities as lipid peroxidation(LPO), reduced glutathione(GSH), superoxide dismutase(SOD) and catalase(CAT) were measured in rat liver homogenate. The biochemical observations were supplemented by histopathological examination.Results:The results demonstrated that treatment withS.xanthocarpumsignificantly(P<0.05-P<0.001) and dose-dependently prevented drug induced increase in serum levels of hepatic enzymes. Furthermore,S. xanthocarpumsignificantly(up toP<0.001) reduced the LPO in the liver tissue and restored activities of defence antioxidant enzymes GSH, SOD and CAT towards normal levels. Histopathology of the liver tissue showed that S. xanthocarpumattenuated the hepatocellular necrosis and led to reduction in inflammatory cells infiltration.Conclusions:The results of this study strongly indicate the protective effect of S. xanthocarpumagainst liver injury which may be attributed to its hepatoprotective activity, and thereby scientifically support its traditional use.

Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of Crohn’s disease

AIM： To investigate overlapping regions of the rpoB gene previously involved with rifamycin resistance in M. tuberculosis and seek correlation between rpoB mutations in dinical MAP strains with susceptibility to RIF and RFB. METHODS： We designed a molecular-based PCR method for the evaluation of rifabutin （RFB） and rifampicin （RIF） resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration （MIC） for RIF was also determined against 11 MAP isolates in attempt to seek correlation with rpoB sequences. RESULTS： We determined that MAP strain 18 had an MIC of 〉 30 mg/L and ≤ 5 mg/L for RIF and RFB respectively, and a significant and novel rpoB mutation C1367T, compared to an MIC of ≤ 1.0 mg/L for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug＇s binding site. In addition, MAP strain 185 contained five silent rpoB mutations and exhibited an MIC comparable to the wild-type. Moreover, our in vitro selected mutation in MAP strain UCF5 resulted in the generation of a new resistant strain （UCF5-RIF16r） that possessed T1442C rpoB mutation and an MIC 〉 30 mg/L and 〉 10 mg/L for RIF and RFB respectively. Sequencing of the entire rpoB gene in MAP strains UCF4, 18, and UCF5-RIF16r revealed an rpoB mutation A2284C further downstream of the 81 bp variable region in UCF4, accounting for observed slight increase in MIC. In addition, no other significant mutations were found in strains 18 and UCF-RIF16r. CONCLUSION： The data clearly illustrates that clinical and in vitro-selected MAP mutants with rpoB mutations result in resistance to RIF and RFB, and that a single amino acid change in the beta subunit may have a significant impact on RIF resistance. Unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections.

Rifampicin inhibits apoptosis in rotenone-induced differentiated PC12 cells by ameliorating mitochondrial oxidative stress

BACKGROUND： Previous studies have shown that rifampicin exhibits neuroprotective effects, but the precise mechanisms remain unclear. Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical scavenger. OBJECTIVE： To investigate the protective effects of rifampicin pretreatment on rotenone-induced mitochondrial oxidative stress in differentiated PC12 cells.DESIGN, TIME AND SETrlNG： A repeated measure, cell-based study was performed at the Department of Neurology, Second Affiliated Hospital, Sun Yat-sen University, China between December 2007 and November 2008. MATERIALS： PC12 cells were a kind gift from the Physiology Laboratory of Zhongshan Medical School, Sun Yat-sen University, China. Rotenone and rifampicin were purchased from Sigma, USA. METHODS： PC12 cells were differentiated by culturing with 100 ng/mL 7S nerve growth factor for 9 days in Dulbecco＇s modified Eagle＇s medium/Nutrient Mix F12 （DMEM/F12） supplemented with 10% fetal bovine serum. The cells were assigned to six groups according to various treatment conditions： control, cultured with normal media; rifampicin group, treated with 300 pmol/L rotenone for 26 hours; rotenone group, treated with 2.5 pmol/L rotenone for 24 hours; rifampicin pretreatment groups, pretreated with 100, 200, and 300 pmol/L rifampicin for 2 hours, respectively, followed by 2.5 μmol/L rotenone for 24 hours.MAIN OUTCOME MEASURES： Mitochondrial membrane potential was measured by fluorescence microscopy and flow cytometry, respectively, using rhodamine123 staining. Intracellular reactive oxygen species formation was analyzed by flow cytometry using 2＇, 7＇-dichlorofluorescin-diacetate staining, and intracellular reduced glutathione was measured with a microplate reader. Cell viability was determined by 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide assay. Cell apoptosis was detected by Hoechst 33342 staining and flow cytometry. RESULTS： Increased apoptosis in rotenone-induced, differentiated, PC12 cells was accompanied by the loss of mitochondrial transmembrane potential, the formation of reactive oxygen species, and reduced glutathione depletion （P 〈 0.01）. Rotenone-induced mitochondrial dysfunction was blocked in a dose-dependent manner by rifampicin （P 〈 0.05 or P 〈 0.01）, CONCLUSION： Pretreatment of differentiated PC12 cells with rifampicin blocked rotenone-induced apoptosis by ameliorating mitochondrial dysfunction and oxidative stress.

Application of Near Infrared Diffuse Reflectance Spectroscopy with Radial Basis Function Neural Network to Determination of Rifampincin Isoniazid and Pyrazinamide Tablets

Partial least squares（PLS）,back-propagation neural network（BPNN）and radial basis function neural network（RBFNN）were respectively used for estalishing quantative analysis models with near infrared（NIR）diffuse reflectance spectra for determining the contents of rifampincin（RMP）,isoniazid（INH）and pyrazinamide（PZA）in rifampicin isoniazid and pyrazinamide tablets.Savitzky-Golay smoothing,first derivative,second derivative,fast Fourier transform（FFT）and standard normal variate（SNV）transformation methods were applied to pretreating raw NIR diffuse reflectance spectra.The raw and pretreated spectra were divided into several regions,depending on the average spectrum and RSD spectrum.Principal component analysis（PCA）method was used for analyzing the raw and pretreated spectra in different regions in order to reduce the dimensions of input data.The optimum spectral regions and the models＇ parameters were chosen by comparing the root mean square error of cross-validation（RMSECV）values which were obtained by leave-one-out cross-validation method.The RMSECV values of the RBFNN models for determining the contents of RMP,INH and PZA were 0.00288,0.00226 and 0.00341,respectively.Using these models for predicting the contents of INH,RMP and PZA in prediction set,the RMSEP values were 0.00266,0.00227 and 0.00411,respectively.These results are better than those obtained from PLS models and BPNN models.With additional advantages of fast calculation speed and less dependence on the initial conditions,RBFNN is a suitable tool to model complex systems.

Effects of four Indian medicinal herbs on Isoniazid-, Rifampicin- and Pyrazinamide-induced hepatic injury and immunosuppression in guinea pigs

AIM: To evaluate and compare the hepatoprotective and immunomodulatory effects of Curcuma longa (CL), Ocimum sanctum (OS), Tinospora cordifolia (TC) and Zizyphus mauritiana (ZM) on liver injury and immunosuppression induced by Isoniazid (INH), Rifampicin (RIF) and Pyrazinamide (PZA). METHODS: Duncan Hartley guinea pigs, weighing 700-1050 g, were treated orally with 50 mg/kg of INH, 100 mg/kg of RIF and 300 mg/Kg of PZA for 21-d. 200 mg/kg (bw) of each herb crude extract was administered to the herb control group and 2-h previous to INH + RIF + PZA (AKT) doses to the Herb + AKT groups. Serum alanine aminotransferase (ALT), aspertate aminotransferase (AST) bilirubin and Alkaline Phosphatase (ALP) were assessed on d 0 and 21 in all the groups. Phagocytic % (P%), Phagocytic Index (PI) and Chemotactic Index (CI) were also measured as immunologic parameters. Histological analysis was carried out to assess injury to the liver. RESULTS: The AKT treated control group showed hepatotoxicity as judged by elevated serum AST 5-fold, AST/ALT ratio 4-fold, ALP 2-fold and hepatological changes, such as focal necrosis, portal triaditis and steatosis. Immune function was suppressed as judged by decreased P% (51.67 ?1.68 vs 40.61 ?1.28, P < 0.01), PI (2.0725±0.05 vs 0.61±0.05, P < 0.001) and CI (1.8525±0.04 vs 0.695±0.07, P < 0.001). All four herb treated groups showed normal liver histology, enzyme levels and increased P%, while PI and CI were enhanced in the TC and ZM treated groups, respectively. CL + AKT, TC + AKT and ZM + AKT showed nearly normal histology with minimal inflammation and microvesicular steatosis, while OS + AKT showed partial protection. Hepatotoxicity was prevented by restricting the rise of AST by 2-fold in CL + AKT and TC + AKT groups and by 3-fold in OS + AKT and ZM + AKT groups, AST/ALT by 2-fold and ALP to normal levels in all four groups. All four herb + AKT groups showed normal to enhanced neutrophil function. CONCLUSION: All four herbs showed hepatoprotective potential and prevented immunosuppression. CL and TC showed the highest hepatoprotective activity, while TC and ZM showed strong immunostimulatory activity.