Ramp Research Articles

Dual Rifampicin and Isoniazid Mannose-Decorated Lipopolysaccharide Nanospheres for Macrophage- Targeted Lung Delivery.

Currently, the treatment protocols for tuberculosis (TB) have several challenges, such as inconsistent oral bioavailability, dose-related adverse effects, and off-target drug toxicity. This research reports the design and characterization of rifampicin (RIF) and isoniazid (INH) loaded hybrid lipid-polysaccharide nanoparticles using the solvent injection method, and demonstrated the influence of conjugated mannosyl residue on macrophage targeting and intracellular drug delivery capacity. The nanospheres, herein called mannose-decorated lipopolysaccharide nanoparticles, were spherical in shape, exhibiting average sizes less than 120 nm (PDI<0.20) and positive zeta potentials. Drug encapsulation was greater than 50% for rifampicin and 60% for isoniazid. The pH-responsive drug release was sustained over a 48-hour period and preferentially released more rifampicin/isoniazid in a simulated acidic phagolysosomal environment (pH 4.8) than in a simulated physiological medium. TGA and FTIR analysis confirmed successful mannose-grafting on nanoparticle surface and optimal degree of mannosylation was achieved within 48-hour mannose-lipopolysaccharide reaction time. The mannosylated nanoparticles were biocompatible and demonstrated a significant improvement towards uptake by RAW 264.7 cells, producing higher intracellular RIF/INH accumulation when compared to the unmannosylated nanocarriers. Overall, the experimental results suggested that mannose-decorated lipopolysaccharide nanosystems hold promise towards safe and efficacious macrophage-targeted delivery of anti-TB therapeutics.

Histopathological and histomorphometric investigation of the effects of different irrigation solutions on Achilles tendon healing in rats.

This study aimed to examine the effect of irrigation fluids containing povidone-iodine (PVP-I), rifampicin (RF), and chlorhexidine gluconate (CHG) used during surgery on healing on a rat Achilles tendon model. Twenty-eight male Sprague-Dawley rats (range, 300 to 400 g) were used in the experiment carried out between November 2022 and December 2022. The rats were divided into PVP-I, RF, CHG, and control groups, with seven rats in each group. Following the tenotomy and repair of the right Achilles tendon, the surgical site was irrigated using PVP-I, RF, CHG, or normal saline (the control group) for 2 min. All rats were sacrificed on the 21st postoperative day. The samples were evaluated histomorphometrically using the scoring system modified by Svensson, Soslowsky, and Cook and histopathologically using the Bonar and Movin classifications. The RF group gave better results in all three scoring systems compared to the control, PVP-I, and CHG groups (p=0.008, p=0.002, and p=0.006, respectively). Cellularity, rounding, and tenocyte morphology showed a significant difference in favor of the RF group (p=0.004). While the distribution of ground substance glycosaminoglycans showed a significant difference in favor of the RF group, there was no significant difference among the other groups (p=0.22). Irrigation solutions containing PVP-I, RF, or CHG show no negative effect on Achilles tendon healing. Moreover, the findings suggest that RF irrigation can accelerate the healing process.

Interactive effect of oral anti-hyperglycaemic or anti-hypertensive drugs on the inhibitory and bactericidal activity of first line anti-TB drugs against M. tuberculosis.

Co-existence of life style disorders, like, Diabetes or Hypertension, increases risk of, treatment failure, deaths and developing drug-resistant TB. Concomitant administration of drugs to treat dual/multi-morbidities may alter their effectiveness, in additive/synergistic or adverse/antagonistic manner. We evaluated interactive effect of 7 anti-hyperglycaemic (HG) and 6 anti-hypertensive (HT) drugs on the inhibitory (MICs) and bactericidal (% killing of intracellular bacilli) activities of anti-TB drugs, Isoniazid (INH), Rifampicin (RFM), Ethambutol (EMB) and Streptomycin (STR) against M. tuberculosis. Five anti-HG drugs, namely, Acarbose, Acetohexamide, Glyburide, Repaglinide and Sitagliptin imparted either 'additive' or 'no effect' on the activities (inhibition or % killing) of all the four anti-TB drugs, as evident by their lower FICs (Fractional Inhibitory concentrations) and higher bacterial killing in combination. Metformin and Rosiglitazone, however, exerted adverse effect on the Ethambutol (FICs >2.0). All the six anti-HT drugs, namely, Atenolol, Hydrochlorothiazide, Ramipril, Valsartan, Nifedipine and Verapamil exerted either 'additive'/'synergistic' or 'no effect' on the activities of anti-TB drugs. These findings may help clinicians to select safe and helpful anti-HG or anti-HT drugs for TB patients, if, suffering with diabetes or hypertension like co-morbidities and receiving DOTs (a set regimen for the treatment of TB based on the WHO guidelines).

Phenotypic drug susceptibility characterization and clinical outcomes of tuberculosis strains with A-probe mutation by GeneXpert MTB/RIF

BackgroundGeneXpert MTB/RIF (Xpert) assay was applied widely to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance.MethodsRetrospectively investigated the association among treatment histories, phenotypic drug susceptibility testing (pDST) results, and clinical outcomes of patients infected with probe A absent mutation isolate confirmed by Xpert.Results63 patients with only probe A absent mutation and 40 with additional pDST results were analyzed. 24 (60.0%) patients had molecular-phenotypic discordant rifampicin (RIF) susceptibility testing results, including 12 (12/13, 92.3%) new tuberculosis (TB) patients and 12 (12/27, 44.4%) retreated ones. 28 (28/39, 71.8%) retreated patients received first-line treatment regime within two years with failed outcomes. New patients had better treatment outcomes than retreated ones (successful: 83.3% VS. 53.8%; P value = 0.02). The clinical results of RIF-susceptible TB confirmed by pDST were not better than RIF-resistant TB (successful: 62.5% VS. 50.0%; P value = 0.43). INH-resistant TB and INH-susceptible TB had similar treatment outcomes too (successful: 61.5% VS. 50.0%; P value = 0.48). 11 (11/12, 91.7%) new patients treated with the short treatment regimen (STR) had successful outcomes.ConclusionsMore than half of mono probe A absent isolates had RIF molecular-phenotypic discordance results, especially in new patients. Probe A mutations were significantly associated with unsuccessful clinical outcomes, whether the pDST results were RIF susceptible or not. STR was the best choice for new patients.Trial registrationretrospectively registered in Wuhan Jinyintan Hospital (No. 2021-KY-16).

A Comparison of the Minimum Inhibitory Concentration of Antibiotics in Staphylococcus Species Isolated From Orthopedic and Respiratory Medicine Infections.

Antibiotic susceptibility is very important for the successful treatment of orthopedic infections, particularly for implant-related infections. While the minimum inhibitory concentrations (MICs) of Staphylococcus species were well investigated for the isolates from the respiratory tract, investigations for orthopedic pathogens are very limited. We investigated the antibiotic MIC values of Staphylococcus species isolated from orthopedic infections and compared them with those of respiratory medicine isolatesused as a control. The MICs of vancomycin (VCM), arbekacin (ABK), teicoplanin (TEIC), linezolid (LZD), and rifampicin (RFP) of a total of consecutive 259 (89 orthopedic and 170 respiratory) Staphylococcus speciesisolated in our laboratory from January 2013 to July 2016 were retrospectively reviewed. Differences between the MICs of each antibiotic in orthopedic and respiratory samples were determined. The number of methicillin-sensitive Staphylococcus aureus (MSSA) with a VCM MIC of <0.5 μg/mL among respiratory isolates was significantly higher than that among orthopedic isolates, while those with a MIC of 2 μg/mL were significantly lower (P = 0.0078). The proportion of methicillin-resistant coagulase-negative staphylococci (MRCNS) isolates with a VCM MIC of 2 μg/mL was significantly higher in orthopedic samples than that of methicillin-resistant Staphylococcus aureus (MRSA) (P < 0.001) in respiratory isolates. The proportion of MRCNS orthopedic isolates with an RFP MIC of >2 μg/mL was significantly higher (P = 0.0058) than that of other orthopedic staphylococci. The VCM MICs of Staphylococcus species from orthopedic infections were higher than those of respiratory samples, particularly MRCNS from implant-related samples.

Minimal effective dose of bedaquiline administered orally and activity of a long acting formulation of bedaquiline in the murine model of leprosy.

Bedaquiline (BDQ), by targeting the electron transport chain and having a long half-life, is a good candidate to simplify leprosy treatment. Our objectives were to (i) determine the minimal effective dose (MED) of BDQ administered orally, (ii) evaluate the benefit of combining two inhibitors of the respiratory chain, BDQ administered orally and clofazimine (CFZ)) and (iii) evaluate the benefit of an intramuscular injectable long-acting formulation of BDQ (intramuscular BDQ, BDQ-LA IM), in a murine model of leprosy. To determine the MED of BDQ administered orally and the benefit of adding CFZ, 100 four-week-old female nude mice were inoculated in the footpads with 5x103 bacilli of M. leprae strain THAI53. Mice were randomly allocated into: 1 untreated group, 5 groups treated with BDQ administered orally (0.10 to 25 mg/kg), 3 groups treated with CFZ 20 mg/kg alone or combined with BDQ administered orally 0.10 or 0.33 mg/kg, and 1 group treated with rifampicin (RIF) 10 mg/kg. Mice were treated 5 days a week during 24 weeks. To evaluate the benefit of the BDQ-LA IM, 340 four-week-old female swiss mice were inoculated in the footpads with 5x103 to 5x101 bacilli (or 5x100 for the untreated control group) of M. leprae strain THAI53. Mice were randomly allocated into the following 11 groups treated with a single dose (SD) or 3 doses (3D) 24h after the inoculation: 1 untreated group, 2 treated with RIF 10 mg/kg SD or 3D, 8 treated with BDQ administered orally or BDQ-LA IM 2 or 20 mg/kg, SD or 3D. Twelve months later, mice were sacrificed and M. leprae bacilli enumerated in the footpad. All the footpads became negative with BDQ at 3.3 mg/kg. The MED of BDQ administered orally against M. leprae in this model is therefore 3.3 mg/kg. The combination of CFZ and BDQ 10-fold lower than this MED did not significantly increase the bactericidal activity of CFZ. The BDQ-LA IM displayed similar or lower bactericidal activity than the BDQ administered orally. We demonstrated that the MED of BDQ administered orally against M. leprae was 3.3 mg/kg in mice and BDQ did not add significantly to the efficacy of CFZ at the doses tested. BDQ-LA IM was similar or less active than BDQ administered orally at equivalent dosing and frequency but should be tested at higher dosing in order to reach equivalent exposure in further experiments.

Molecular Detection of Multidrug Resistance and Characterizations of Mutations in Mycobacterium Tuberculosis Using Polycarbonate Track-Etched Membrane Based DNA Bio-Chip.

With the widespread use of rifampicin (RMP) and isoniazid (INH), multidrug resistance (MDR) in Mycobacterium tuberculosis (M.tb) poses a threat to the success of tuberculosis (TB) control programs. We have developed a new polycarbonate track-etched membranes(PC-TEM) based DNA bio-chip designed for rapid detection of mutations conferring MDR inM.tbculture isolates. Bio-chips were designed to contain 14 specific probes for wild type and mutated allele of selected codons within 80bprifampicin resistance determining region of rpoB gene, katGgene andmabA-inhAregulatory region. RMP-resistance-associated gene mutation pointsrpoB 516, 526, 531and533, and the INH-resistance-associated gene mutation pointskatG315andinhA-15were targeted. Bio-chip signal was detected using enhanced chemiluminescence. A total of 50 culture isolates that were sensitive or resistant to RMP and/or INH were analyzed by bio-chip. The results of culture-based drug susceptibility testing (DST)were used as the gold standard and gene sequencing was performed to resolve the discordance. Amongst 50 culture isolates, we have detected 18 MDR, 9 RMP mono-resistant, 6 INH mono-resistant, and 17 fully susceptible isolates. The developed DNA bio-chip has a sensitivity of 90% for RMP and MDR and 100% for INH resistance. The bio-chip has a specificity of 100% for RMP and MDR and 88.8% for INH detection. The identification of mutations using the DNA bio-chip was 100% concordant with the sequencing data for the probes covered by the bio-chip. The detection ofrpoB, katGand inhA gene mutation points by a DNA bio-chip may be used as a rapid, accurate, and economical, clinical detection method for MDR detection in M.tb. This is very valuable for the control of TB epidemics.

Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.

Characterizing Neisseria meningitidis in Southern Vietnam between 2012 and 2021: A predominance of the chloramphenicol-resistant ST-1576 lineage

ObjectivesOur goal was to describe Invasive Meningococcal Disease (IMD) in Southern Vietnam over the last 10 years. We characterized 109 Neisseria meningitidis strains in Southern Vietnam isolated between 1980s to 2021, that were collected from IMD (n = 44), sexually transmitted infections (n = 2), and healthy carriage (n = 63). MethodsIMD were confirmed by bacterial culture and/or real-time polymerase chain reaction at the national reference laboratory in Pasteur Institute of Ho Chi Minh City (PIHCM). Antimicrobial resistance was determined on 31 IMD and two sexually transmitted infection isolates with E-test for chloramphenicol (CHL), penicillin (PEN), ciprofloxacin (CIP), ceftriaxone (CRO), and rifampicin (RIF). Sequencing was performed for analyzing of multilocus-sequence-typing (MLST), porA, fetA, and antibiotic resistance genes, including gyrA, penA, and rpoB. ResultsThe incidence rate during this period was 0.02 per 100,000 persons/year. Serogroup B accounted for over 90% of cases (50/54). ST-1576 were mainly responsible for IMD, 27/42 MLST profiles, and associated with CHL resistance. Resistance was prevalent among IMD isolates. Thirteen were resistant to CHL (minimum inhibitory concentration [MIC] ≥16 mg/l), 12 were intermediate to PEN (MIC between 0.19 and 0.5 mg/l), and five were CIP-resistant (MIC between 0.19 and 0.5 mg/l). Particularly, one was non-susceptible to CRO (MIC at 0.125 mg/l), belonging to ST-5571 lineage. The resistance was due to carrying resistant alleles of penA and gyrA genes, and catP gene. Notably, seven isolates were resistant/non-susceptible to two or more antibiotics. ConclusionOur results suggest the persistence of the circulating ST-1576 in Southern Vietnam, with a spread of antimicrobial resistance across the community.

Population pharmacokinetic model of rifampicin for personalized tuberculosis pharmacotherapy: Effects of SLCO1B1 polymorphisms on drug exposure

Rifampicin (RIF) exhibits high pharmacokinetic (PK) variability among individuals; a low plasma concentration might result in unfavorable treatment outcomes and drug resistance. This study evaluated the contributions of non- and genetic factors to the interindividual variability of RIF exposure, then suggested initial doses for patients with different weight bands. This multicenter prospective cohort study in Korea analyzed demographic and clinical data, the solute carrier organic anion transporter family member 1B1 (SLCO1B1) genotypes, and RIF concentrations. Population PK modeling and simulations were conducted using nonlinear mixed-effect modeling. In total, 879 tuberculosis (TB) patients were divided into a training dataset (510 patients) and a test dataset (359 patients). A one-compartment model with allometric scaling for effect of body size best described the RIF PKs. The apparent clearance (CL/F) was 16.6% higher among patients in the SLCO1B1 rs4149056 wild-type group than among patients in variant group, significantly decreasing RIF exposure in the wild-type group. The developed model showed better predictive performance compared with previously reported models. We also suggested that patients with body weights of <40 kg, 40-55 kg, 55-70 kg, and >70 kg patients receive RIF doses of 450, 600, 750, and 1050 mg/day, respectively. Total body weight and SLCO1B1 rs4149056 genotypes were the most significant covariates that affected RIF CL/F variability in Korean TB patients. We suggest initial doses of RIF based on World Health Organization weight-band classifications. The model may be implemented in treatment monitoring for TB patients.

Performance of the MeltPro TB assay as initial test for diagnosis of pulmonary tuberculosis with drug-resistance detection

BackgroundThe MeltPro TB assay (MeltPro) is a molecular rapid diagnostic test designed for detecting resistance to antituberculosis drugs. However, the performance of MeltPro as an initial diagnostic test for simultaneously detecting the presence of Mycobacterium tuberculosis (MTB) and drug resistance has not been evaluated. This study aims to assess the performance of MeltPro as initial diagnostic test for simultaneous detection of MTB and drug resistance in clinical samples from patients with presumptive pulmonary tuberculosis (PTB).MethodsA retrospective analysis was conducted on 1283 patients with presumptive PTB from two clinical centers, out of which 875 were diagnosed with PTB. The diagnostic accuracy of MeltPro, Xpert MTB/RIF (Xpert), and MGIT 960 for PTB detection was evaluated. Rifampicin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (STR), and fluoroquinolone (FQ) resistance were detected using MeltPro, with Xpert and/or the broth microdilution plate method (MYCOTB) results as references.ResultsFor the diagnosis of PTB, MeltPro showed a sensitivity of 69.0%, which was similar to Xpert (72.7%; P > 0.05) and higher than MGIT (58.1%; P < 0.001). The specificity of MeltPro was 97.1%, similar to Xpert (98.0%; P > 0.05). In smear-negative patients, MeltPro's sensitivity was 50.9%, similar to Xpert (56.5%; P > 0.05), and higher than MGIT (33.1%; P < 0.001). Based on Xpert and/or MYCOTB results, MeltPro exhibited a sensitivity and specificity of 98.3% and 99.2%, respectively, for detecting RIF resistance. Based on MYCOTB results, MeltPro's sensitivity for detecting resistance to INH, EMB, STR, and FQ was 96.4%, 89.1%, 97.5%, and 90.3%, respectively, with specificities of 96.0%, 96.0%, 95.2%, and 99.4%, respectively.ConclusionThe MeltPro TB assay could potentially be an effective alternative as the initial test for rapid diagnosis of PTB with drug-resistance detection in clinical practice.

Toxin-antitoxin Genes Expression in Multidrug-Resistant Mycobacterium tuberculosis Isolates under Drug Exposure.

Toxin-antitoxin systems (TAs) are highly conserved in Mycobacterium tuberculosis (Mtb). The TAs role in maintaining and disseminating drug resistance in bacterial populations has been indicated. So, we aimed to analyze the expression level of MazEF-related genes in drug-susceptible and multi drug-resistant (MDR) Mtb isolates under isoniazid (INH) and rifampin (RIF) stress. We obtained 23 Mtb isolates, including 18 MDR and 5 susceptible isolates, from the Ahvaz Regional TB Laboratory collection. The expression level of mazF3, mazF6, mazF9 toxin genes and mazE3, mazE6, mazE9 antitoxin genes in MDR and susceptible isolates was evaluated by quantitative real-time PCR (qRT-PCR) after exposure to RIF and INH. The mazF3, F6, and F9 toxin genes were overexpressed in at least two MDR isolates in the presence of RIF and INH, in contrast to mazE antitoxin genes. More MDR isolates were induced to overexpress mazF genes by RIF than INH (72.2% vs. 50%). Compared to the H37Rv strain, and susceptible isolates, the expression levels of mazF3,6 by RIF and mazF3,6,9 by INH were significantly upregulated in MDR isolates (p<0.05) but no remarkable difference was detected in the expression level of mazF9 genes by INH between these groups. In susceptible isolates, the expression levels of mazE3,6 by RIF and mazE3,6,9 by INH were induced and enhanced significantly compared with MDR isolates, but there was no difference between MDR and H37Rv strain. Based on the results, we propose that mazF expression under RIF/INH stress may be associated with drug resistance in Mtb in addition to mutations, and the mazE antitoxins may be related to enhanced susceptibility of Mtb to INH and RIF. Further experiments are needed to investigate the exact mechanism underlying the TA system role in drug resistance.

Drug resistance and the genotypic characteristics of rpoB and katG in rifampicin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates in central Vietnam.

Tuberculosis (TB) and drug-resistant TB (DR-TB) are national health burdens in Vietnam. In this study, we investigated the prevalence of rifampicin (RIF) and/or isoniazid (isonicotinic acid hydrazide, INH) resistance in patients with suspected TB, and applied appropriate techniques to help rapidly target DR-TB. In total, 1,547 clinical specimens were collected and cultured using the BACTEC MGIT system (Becton Dickinson and Co.). A resazurin microtiter assay (REMA) was used to determine the proportions of RIF and/or INH resistance. A real-time polymerase chain reaction panel with TaqMan probes was employed to identify the mutations of rpoB and katG associated with DR-TB in clinical isolates. Genotyping of the identified mutations was also performed. A total of 468 Mycobacterium tuberculosis isolates were identified using the REMA. Of these isolates, 106 (22.6%) were found to be resistant to 1 or both antibiotics. Of the resistant isolates, 74 isolates (69.8%) were resistant to isoniazid (INH) only, while 1 isolate (0.94%) was resistant to RIF only. Notably, 31 isolates (29.24%) were resistant to both antibiotics. Of the 41 phenotypically INH-resistant isolates, 19 (46.3%) had the Ser315Thr mutation. There were 8 different rpoB mutations in 22 (68.8%) of the RIF-resistant isolates. The most frequently detected mutations were at codons 531 (37.5%), 526 (18.8%), and 516 (6.3%). To help prevent new cases of DR-TB in Vietnam, it is crucial to gain a comprehensive understanding of the genotypic DR-TB isolates.

Spectroscopy and dynamics of beta-lactoglobulin complexed with rifampicin

In this paper, we report the binding interaction of milk protein, beta-lactoglobulin (BLG), with an antibiotic against tuberculosis, rifampicin (RIF). BLG intrinsic fluorescence from tryptophan (Trp) amino acids was monitored to understand protein-drug interactions. Binding parameters and stoichiometry were estimated with the help of fluorescence spectral changes. Synchronous fluorescence spectroscopy was employed to exclusively monitor the Trp and Tyrosine (Tyr) environment in the presence of RIF. With the help of steady state fluorescence at different temperatures supported by time-resolved fluorescence, we confirmed that the protein forms a static complex with RIF. Thermodynamic parameters, ΔH and ΔS values, showed the involvement of hydrophobic forces between the RIF and BLG. Competitive displacement assay with ANS confirmed the BLG calyx as the binding site for RIF. Energy transfer mechanism from Trp to RIF was attributed to the fluorescence changes in protein upon complexation. The Förster resonance energy transfer (FRET) was used to find distance, energy transfer efficiency and rate of energy transfer between donor (BLG) and acceptor (RIF). Fourier-transform infrared (FTIR) spectroscopy was utilized for estimating changes in the secondary structure of BLG induced by RIF. Molecular docking was used to visualise the binding location of RIF on BLG. Molecular dynamics (MD) simulation studies showed a consistent binding interactions between BLG and RIF during the 100 ns simulation period and this well supported the increased beta sheet content in FTIR. Overall our results establish the potential of intrinsic fluorescence of BLG in combination with biophysical tools to rationalize drug-protein interactions. Communicated by Ramaswamy H. Sarma

Hepatoprotective effect of hydromethanol extract of Otostegia integrifolia benth leaves in isoniazid and rifampicin induced Swiss albino mice

IntroductionDrug-induced liver injury is the most common cause of acute liver failure. Off-Target effect “hepatotoxicity “frequently detected during clinical examination of patients on anti-Tb medication particularly isoniazid (INH), and rifampin (RMP). However, there is no any treatment option against isoniazid and rifampicin induced hepatotoxicity. It is, therefore, necessary to search for effective affordable and safe drugs from medicinal plants for the prevention of liver toxicity caused by isoniazid and rifampicin. The aim the current study is to evaluate hepatoprotective effect of hydro methanol extract from Otostegia integrifolia leaves in isoniazid and rifampicin-induced hepatotoxicity in Swiss albino mice. MethodsO. integrifolia leaves powder was macerated in hydromethanol and thirty Swiss albino mice 29.0–40.6 g were grouped in to five groups. Group I were given 20 ml/kg distilled water, group II were given 100 mg INH and 150 mg RIF per kg body weight. Group III, group IV, and group V were given 200 mg extract, 400 mg extract, and 100 mg of N-acetyl cysteine respectively per kg 1hr before induction with 100 mg INH plus 150 mg RIF per kg. The treatments were followed for 14 days. On the 15th day, all mice were anaesthetized with diethyl ether; blood samples were collected for the assessment liver enzyme and function test. ResultsGroup II mice's serum ALT, AST and total bilirubin levels were significantly increased and serum total protein and albumin levels were significantly decreased as compared with group I mice. The groups of mice treated with O. integrifolia at a dose of 400 mg/kg and N-acetyl cysteine AST, ALT and total bilirubin level were significantly decreased; and total protein and albumin levels were significantly (P < 0.05) increased as compared with group II. The liver index of the group IV showed decreased (P < 0.05) as compared to the group II. ConclusionEvidence from our study revealed that the hydromethanol extract of O. integrifolia has a hepatoprotective effect against isoniazid and rifampicin-induced hepatotoxicity in Swiss Albino mice. This protective effect of O. integrifolia extract may be based on its metal ion reducing power, free radical scavenging activity, and anti-inflammatory activity and could be used as a potential therapeutic option.

Combinatorial efficacy of Manuka honey and antibiotics in the in vitro control of staphylococci and their small colony variants

IntroductionStaphylococci are among the list of problematic bacteria contributing to the global antibiotic resistance (ABR) crisis. Their ability to adopt the small colony variant (SCV) phenotype, induced by prolonged antibiotic chemotherapy, complicates staphylococcal infection control options. Novel and alternative approaches are needed to tackle staphylococcal infections and curb ABR. Manuka honey (MH), a non-antibiotic alternative is recognized for its unique antibacterial activity based on its methylglyoxal (MGO) component.MethodsIn this study, MH (MGO 830+) was tested in combination with gentamicin (GEN), rifampicin (RIF), or vancomycin (VA) against staphylococcal wildtype (WT) and SCVs. To our knowledge, there are no current studies in the literature documenting the effects of MH on staphylococcal SCVs. While Staphylococcus aureus is well-studied for its international ABR burden, limited data exists demonstrating the effects of MH on S. epidermidis and S. lugdunensis whose pathogenic relevance and contribution to ABR is also rising.Results &amp;amp; discussionThe three staphylococci were most susceptible to RIF (0.06-0.24 μg/ml), then GEN (0.12-0.49 μg/ml), and lastly VA (0.49-0.96 μg/ml). The MICs of MH were 7%, 7-8%, and 6-7% (w/v), respectively. Fractional inhibitory concentration (FIC) evaluations showed that the combined MH + antibiotic effect was either additive (FICI 1-2), or partially synergistic (FICI &amp;gt;0.5-1). While all three antibiotics induced SCVs in vitro, stable SCVs were observed in GEN treatments only. The addition of MH to these GEN-SCV-induction analyses resulted in complete suppression of SCVs (p&amp;lt;0.001) in all three staphylococci, suggesting that MH’s antibacterial properties interfered with GEN’s SCV induction mechanisms. Moreover, the addition of MH to growth cultures of recovered stable SCVs resulted in the inhibition of SCV growth by at least 99%, indicating MH’s ability to prevent subsequent SCV growth. These in vitro analyses demonstrated MH’s broad-spectrum capabilities not only in improving WT staphylococci susceptibility to the three antibiotics, but also mitigated the development and subsequent growth of their SCV phenotypes. MH in combination with antibiotics has the potential to not only resensitize staphylococci to antibiotics and consequently require less antibiotic usage, but in instances where prolonged chemotherapy is employed, the development and growth of SCVs would be hampered, providing a better clinical outcome, all of which mitigate ABR.

Evaluation of different diagnostic methods for spinal tuberculosis infection

Background and purposeTuberculosis (TB) is the most fatal infectious disease worldwide. Approximately 24.6% of tuberculosis cases are extrapulmonary and predominantly affect the spine. It is difficult to diagnose spinal TB (STB). We aimed to evaluate the diagnostic performance of the Mycobacteria Growth Indicator Tube (MGIT)-960 culture, T-SPOT.TB, Xpert Mycobacterium tuberculosis complex (MTB)/resistance to rifampin (RIF), and Metagenomic Next-Generation Sequencing (mNGS) to detect STB.MethodsWe assessed 126 patients presumed to have STB using these four methods. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using clinical diagnosis as a reference.ResultsOf the patients, 41 were diagnosed with STB and 85 with non-STB. In the STB group, the sensitivity, specificity, PPV, and NPV of the MGIT-960 culture were 29.3% (12/41), 100% (85/85), 100% (12/12), and 74.6% (85/114), respectively. The sensitivity, specificity, PPV, and NPV of T-SPOT.TB were 92.7% (38/41), 82.4% (70/85), 58.5% (31/53), and 95.9% (70/73), respectively. The sensitivity, specificity, PPV, and NPV of the Xpert MTB/RIF assay were 53.7% (22/41), 100% (85/85), 100% (22/22), and 81.7% (85/104), respectively. The sensitivity, specificity, PPV, and NPV of mNGS were 39.0% (16/41), 98.8% (84/85), 94.1% (16/17), and 77.1% (84/109), respectively. The sensitivity, specificity, PPV, and NPV of mNGS + Xpert MTB/RIF were 73.2% (30/41), 100% (85/85), 96.8% (30/31), and 72.0% (85/118), respectively. The sensitivity, specificity, PPV, and NPV of the mNGS + T-spot assay were 97.6% (40/41), 100% (85/85), 67.9% (38/56), and 75.9% (85/113), respectively. Moreover, the sensitivity, specificity, PPV, and NPV of T-spot + Xpert MTB/RIF were 95.1% (39/41), 100% (85/85), 72.2% (39/54), and 81.0% (85/105), respectively.ConclusionsT-SPOT.TB is the most effective method for diagnosing STB; however, Xpert MTB/RIF is more reliable and can detect RIF resistance. Clinicians can use mNGS to identify pathogens in patients with spinal infections; these pathogens appeared to be more meaningful in guiding the clinical management of patients in the non-STB group. The combination of Xpert MTB/RIF and mNGS can improve the early diagnosis rate and drug resistance detection, reduce the diagnostic cycle, and provide early targeted anti-TB treatment for patients with STB.

Diagnostic Utility of Cartridge-Based Nucleic Acid Amplification Test (CBNAAT) on Induced Sputum Versus Gastric Aspirate Samples for the Diagnosis of Paediatric Pulmonary Tuberculosis.

Tuberculosis (TB) in children is neglected, mainly due to a lack of sensitive diagnostic tools. Paediatric TB is now a global priority. More paediatric TB cases are being recorded as a result of the introduction of Xpert® Mycobacterium tuberculosis (MTB)/rifampicin (RIF) (Cepheid Inc., Sunnyvale, USA). This study was undertaken to evaluate the performance of Xpert MTB/RIF in the diagnosis of pulmonary TB in children. We recruited 70 paediatric patients with probable pulmonary TB and their gastric aspirate (GA), and induced sputum (IS) samples were collected between January 2021 and June 2022 in Saifai, Etawah, Uttar Pradesh, at the Microbiology Department of the Uttar Pradesh University of Medical Sciences (U.P.U.M.S.). All samples were subjected to smear examination, Bacterial Activation ofContinuousTemperature andEnvironmentalControl -MycobacterialGrowthIndicatorTube (BACTEC-MGIT) culture, and Xpert MTB/RIF. The specimens included 70 GAs and 70 IS samples. The total number of specimens were 140 and we collected GA as well as IS from each of the patient enrolled in the study.When compared to microscopy, GeneXpert provides a quicker and earlier detection of paediatric TB. The sensitivity of the cartridge-based nucleic acid amplification test (CBNAAT) against mycobacterial growth indicator tube (MGIT) was 75.0% for GA samples and 63.64% for IS samples. Paediatric TB, owing to its paucibacillary nature and difficulty in the collection of samples, makes the diagnosis difficult by conventional methods. Our study shows that smear and culture yield in GA samples are superior to those of IS samples and the sensitivity of Xpert MTB/RIF assay is also significantly different in GA and IS samples, but a combination of GA and IS yielded the best results.

Comprehensive assessment of invalid and indeterminate results in Truenat MTB-RIF testing across sites under the national TB elimination program of India.

Truenat MTB-RIF assay (Truenat), a nucleic acid amplification test (NAAT), is a real-time polymerase chain reaction (RT-PCR) chip-based assay that can detect Mycobacterium tuberculosis (Mtb) and rifampicin (RIF) drug resistance using portable, battery-operated devices. The National TB Elimination Program (NTEP) in India introduced this novel tool at the district and subdistrict level in 2020. This study aimed to assess the level and causes of inconclusive results (invalid results, errors, and indeterminate results) in MTB and RIF testing at NTEP sites and the root causes of these in the programmatic setting. Truenat testing data from 1,690 functional Truenat sites under the NTEP from April to June 2021 were analyzed to assess the rates of errors, invalid MTB results, and indeterminate RIF results. Following this analysis, 12 Truenat sites were selected based on site performance in Truenat testing, diversity of climatic conditions, and geographical terrain. These sites were visited to assess the root causes of their high and low rates of inconclusive results using a structured checklist. A total of 327,649 Truenat tests performed for MTB and RIF testing were analyzed. The rate of invalid MTB results was 5.2% [95% confidence interval (CI): 5.11-5.26; n = 16,998] and the rate of errors was 2.5% (95% CI: 2.46-2.57; n = 8,240) in Truenat MTB chip testing. For Mtb-positive samples tested using the Truenat RIF chip for detection of RIF resistance (n = 40,926), the rate of indeterminate results was 15.3% (95% CI: 14.97-15.67; n = 6,267) and the rate of errors was 1.6% (95% CI: 1.53-1.78; n = 675). There was a 40.1% retesting gap for Mtb testing and a 78.2% gap for inconclusive RR results. Among the inconclusive results retested, 27.9% (95% CI: 27.23-28.66; n = 4,222) were Mtb-positive, and 9.2% (95% CI: 7.84-10.76; n = 139) were detected as RR. The main causes affecting Truenat testing performance include suboptimal adherence to standard operating procedures (SOPs), inadequate training, improper storage of testing kits, inadequate sputum quality, lack of quality control, and delays in the rectification of machine issues. Root cause analysis identified that strengthening of training, external quality control, and supervision could improve the rate of inconclusive results. Ensuring hands-on training of technicians for Truenat testing and retesting of samples with inconclusive results are major recommendations while planning for Truenat scale-up. The recommendations from the study were consolidated into technical guidance documents and videos and disseminated to laboratory staff working at the tiered network of TB laboratories under the NTEP in order to improve Truenat MTB-RIF testing performance.

Correlations among the plasma concentrations of first-line anti-tuberculosis drugs and the physiological parameters influencing concentrations.

Background: The plasma concentrations of the four most commonly used first-line anti-tuberculosis (TB) drugs, isoniazid (INH), rifampicin (RMP), ethambutol (EMB), and pyrazinamide (PZA), are often not within the therapeutic range. Insufficient drug exposure could lead to drug resistance and treatment failure, while excessive drug levels may lead to adverse reactions. The purpose of this study was to identify the physiological parameters influencing anti-TB drug concentrations. Methods: A retrospective cohort study was conducted. The 2-h plasma concentrations of the four drugs were measured by using the high-performance liquid chromatography-tandem mass spectrometry method. Results: A total of 317 patients were included in the study. The proportions of patients with INH, RMP, EMB, and PZA concentrations within the therapeutic range were 24.3%, 31.5%, 27.8%, and 18.6%, respectively. There were positive associations between the concentrations of INH and PZA and RMP and EMB, but negative associations were observed between the concentrations of INH and RMP, INH and EMB, RMP and PZA, and EMB and PZA. In the multivariate analysis, the influencing factors of the INH concentration were the PZA concentration, total bile acid (TBA), serum potassium, dose, direct bilirubin, prealbumin (PA), and albumin; those of the RMP concentration were PZA and EMB concentrations, weight, α-l-fucosidase (AFU), drinking, and dose; those of the EMB concentration were the RMP and PZA concentrations, creatinine, TBA and indirect bilirubin; and those of the PZA concentration were INH, RMP and EMB concentrations, sex, weight, uric acid and drinking. Conclusion: The complex correlations between the concentrations of the four first-line anti-TB drugs lead to a major challenge in dose adjustment to maintain all drugs within the therapeutic window. Levels of TBA, PA, AFU, and serum potassium should also be considered when adjusting the dose of the four drugs.