Ceftriaxone efficacy for Mycobacterium avium complex lung disease in the hollow fiber and translation to sustained sputum culture conversion in patients.

BACKGROUND: Only 35.6%-50.8% of patients with Mycobacterium avium complex (MAC) pulmonary disease achieve sustained sputum culture conversion (SSCC) on treatment with the azithromycin-ethambutol-rifabutin standard of care (SOC). We tested the efficacy of ceftriaxone, a ß-lactam with a lung penetration ratio of 12.18-fold. METHODS: We mimicked lung concentration-time profiles of seven ceftriaxone once-daily doses for 28 days in the hollow fiber system model of intracellular MAC (HFS- MAC). Monte Carlo experiments were used for dose selection.We also compared the once-daily ceftriaxone monotherapy to three-drug SOC against five MAC clinical isolates in HFS-MAC using γ (kill)-slopes. Results were translated to SSCC rates. RESULTS: Ceftriaxone killed 1.02-3.82 log10 cfu/mL in dose-response studies. Ceftriaxone 2G once-daily was identified as the optimal dose. Ceftriaxone killed all five strains below day 0 versus 2/5 for SOC. The median γ (95% confidence interval) was 0.49(0.47-0.52) log10 cfu/mL/day for ceftriaxone and 0.38(0.34-0.43) log10 cfu/mL/day for SOC. In patients, the SOC was predicted to achieve SSCC rates of 39.3%(36%-42%) at 6 months (similar to meta-analyses results). The SOC SSCC was 50% at 8.18(3.64-27.66) months versus 3.58(2.20-7.23) months for ceftriaxone. Thus, ceftriaxone shortened time-to-SSCC 2.35-fold compared to SOC. CONCLUSION: Ceftriaxone is a promising agent for creation of short-course chemotherapy.

Nouveau short-course therapy and morphism mapping for clinical pulmonary Mycobacterium kansasii.

Standard therapy [isoniazid, rifampin, ethambutol], with or without a macrolide, for pulmonary Mycobacterium kansasii lasts more than a year. Therefore, shorter treatment duration regimens are required. We used data from 32 Taiwanese patients treated with standard therapy who were followed using repetitive sampling-based sputum Mkn time-to-positivity in liquid cultures to calculate kill slopes [γ] based on ordinary differential equations and time-to-extinction of each patient's bacterial burden. The γ was 0.18 [95% Confidence Interval (CI): 0.16-0.20] log10 CFU/mL/day on standard therapy. Next, we identified Mkn time-to-extinction in the hollow fiber system model of pulmonary M. kansasii disease [HFS-Mkn] treated with standard therapy, which was a γ of 0.60 [95% CI: 0.45-0.69) log10 CFU/mL/day. The γs and time-to-extinctions between the two datasets formed structure-preserving maps based on category theory: thus, we could map them from one to the other using morphisms. This mapping identified a multistep non-linear transformation-factor for time-to-extinction from HFS-Mkn to patients. Next, a head-to-head study in the HFS-Mkn identified median time-to-extinction for standard therapy of 38.7 [95% CI: 29.1-53.2) days, isoniazid-rifampin-ethambutol-moxifloxacin of 21.7 [95% CI: 19.1-25) days, isoniazid-rifampin-moxifloxacin of 22 [96% CI: 20.1-24.5) days, and rifampin-moxifloxacin-tedizolid of 20.7 [95% CI:18.5-29) days. Our transformation-factor based translation predicted the proportion of patients of 90.7 [88.74-92.35)% achieving cure with standard therapy at 12 months, and 6-months cure rates of 99.8 [95% CI: 99.27-99.95)% for isoniazid-rifampin-ethambutol-moxifloxacin, 92.2 [90.37-93.71)% for isoniazid-rifampin-moxifloxacin, and 99.9 [99.44-99.99)% for rifampin-moxifloxacin-tedizolid. Thus, rifampin-moxifloxacin-tedizolid and isoniazid-rifampin-ethambutol-moxifloxacin are predicted to be short-course chemotherapy regimens for pulmonary M. kansasii disease.

Potency of the novel PolC DNA polymerase inhibitor CRS0540 in a disseminated Listeria monocytogenes intracellular hollow-fibre model.

BACKGROUND: Listeriosis is an orphan disease, which is nevertheless fatal in immunocompromised people. CRS0540 is a novel PolC DNA polymerase inhibitor that has demonstrated good in vitro and in vivo activity against Listeria monocytogenes. METHODS: Rodent-to-human allometry projection-based human population pharmacokinetics of CRS0540 were used for all studies. CRS0540 pharmacokinetics/pharmacodynamics studies in an intracellular hollow-fibre system model of disseminated listeriosis (HFS-Lister) examined the effect of eight treatment doses, administered daily over 7 days, in duplicate units. Total bacterial burden versus AUC/MIC exposures on each day were modelled using the inhibitory sigmoid Emax model, while CRS0540-resistant bacterial burden was modelled using a quadratic function. Ten thousand-subject Monte Carlo simulations were used to predict an optimal clinical dose for treatment. RESULTS: The mean CRS0540 intracellular/extracellular AUC0-24 ratio was 34.07 (standard error: 15.70) as measured in the HFS-Lister. CRS0540 demonstrated exposure-dependent bactericidal activity in the HFS-Lister, with the highest exposure killing approximately 5.0 log10 cfu/mL. The free drug AUC0-24/MIC associated with 80% of maximal kill (EC80) was 36.4. Resistance emergence versus AUC/MIC was described by a quadratic function, with resistance amplification at an AUC/MIC of 54.8 and resistance suppression at an AUC/MIC of 119. Monte Carlo simulations demonstrated that for the EC80 target, IV CRS0540 doses of 100 mg/kg achieved PTAs of >90% at MICs up to 1.0 mg/L. CONCLUSIONS: CRS0540 is a promising orphan drug candidate for listeriosis. Future PK/PD studies comparing it with penicillin, the standard of care, could lead to this drug as a new treatment in immunocompromised patients.

Omadacycline efficacy in the hollow fibre system model of pulmonary Mycobacterium avium complex and potency at clinically attainable doses.

OBJECTIVES: The standard of care (SOC) for the treatment of pulmonary Mycobacterium avium complex (MAC) disease (clarithromycin, rifabutin, and ethambutol) achieves sustained sputum conversion rates of only 54%. Thus, new treatments should be prioritized. METHODS: We identified the omadacycline MIC against one laboratory MAC strain and calculated drug half life in solution, which we compared with measured MAC doubling times. Next, we performed an omadacycline hollow fibre system model of intracellular MAC (HFS-MAC) exposure-effect study, as well as the three-drug SOC, using pharmacokinetics achieved in patient lung lesions. Data was analysed using bacterial kill slopes (γ-slopes) and inhibitory sigmoid Emax bacterial burden versus exposure analyses. Monte Carlo experiments (MCE) were used to identify the optimal omadacycline clinical dose. RESULTS: Omadacycline concentration declined in solution with a half-life of 27.7 h versus a MAC doubling time of 16.3 h, leading to artefactually high MICs. Exposures mediating 80% of maximal effect changed up to 8-fold depending on sampling day with bacterial burden versus exposure analyses, while γ-slope-based analyses gave a single robust estimate. The highest omadacycline monotherapy γ-slope was -0.114 (95% CI: -0.141 to -0.087) (r2 = 0.98) versus -0.114 (95% CI: -0.133 to -0.094) (r2 = 0.99) with the SOC. MCEs demonstrated that 450 mg of omadacycline given orally on the first 2 days followed by 300 mg daily would achieve the AUC0-24 target of 39.67 mg·h/L. CONCLUSIONS: Omadacycline may be a potential treatment option for pulmonary MAC, possibly as a back-bone treatment for a new MAC regimen and warrants future study in treatment of this disease.

Potency of vancomycin against Mycobacterium tuberculosis in the hollow fiber system model.

OBJECTIVES: To determine whether an inhaled vancomycin formulation resulting in high intrapulmonary 24-h area under the concentration-time curve (AUC0-24) could be optimised for tuberculosis treatment. We also explored vancomycin synergy and antagonism with d-cycloserine and benzylpenicillin. METHODS: We determined MICs of two Mycobacterium tuberculosis (Mtb) laboratory strains (H37Ra and H37Rv) and two drug-susceptible and nine multidrug resistant clinical strains. Second, in the hollow fiber system model of TB [HFS-TB] using Mtb H37Ra strain, we recapitulated vancomycin intrapulmonary pharmacokinetics of eight doses administered twice daily over 28 days, mimicking a 6-h half-life. Using the HFS-TB, vancomycin was tested in combination with d-cycloserine and benzylpenicillin to determine synergy or antagonism between drugs targeting the same pathway. RESULTS: Vancomycin MICs were 12 and 48 mg/L in drug-susceptible clinical isolates but >96 mg/L in all MDR isolates.In the HFS-TB, vancomycin killed 3.9 ± 0.6 log10 CFU/mL Mtb. The EC50 was calculated as AUC0-24/MIC of 184.6 ± 106.5. Compared with day 0, 1.0 and 2.0 log10 CFU/mL kill was achieved by AUC0-24/MIC of 168 and 685, respectively. Acquired vancomycin resistance developed to all vancomycin doses tested in the HFS-TB. In the HFS-TB, vancomycin was antagonistic to benzylpenicillin, which works downstream to glycopeptides in peptidoglycan synthesis, but synergistic with d-cycloserine, which inhibits upstream d-Ala-d-Ala ligase and alanine racemase. CONCLUSION: Our proof-of-concept studies show that vancomycin optimal exposure target for Mtb kill could be achieved via inhalational drug delivery. Addition of drugs synergistic with vancomycin, e.g. d-cycloserine, may lower the vancomycin concentrations required to kill Mtb.

Cumulative Fraction of Response for Once- and Twice-Daily Delamanid in Patients with Pulmonary Multidrug-Resistant Tuberculosis.

Pharmacokinetic (PK) and pharmacodynamic (PD) analyses were conducted to determine the cumulative fraction of response (CFR) for 100 mg twice-daily (BID) and 200 mg once-daily (QD) delamanid in patients with multidrug-resistant tuberculosis (MDR-TB), using a pharmacodynamic target (PDT) that achieves 80% of maximum efficacy. First, in the mouse model of chronic TB, the PK/PD index for delamanid efficacy was determined to be area under the drug concentration-time curve over 24 h divided by MIC (AUC0-24/MIC), with a PDT of 252. Second, in the hollow-fiber system model of tuberculosis, plasma-equivalent PDTs were identified as an AUC0-24/MIC of 195 in log-phase bacteria and 201 in pH 5.8 cultures. Third, delamanid plasma AUC0-24/MIC and sputum bacterial decline data from two early bactericidal activity trials identified a clinical PDT of AUC0-24/MIC of 171. Finally, the CFRs for the currently approved 100-mg BID dose were determined to be above 95% in two MDR-TB clinical trials. The CFR for the 200-mg QD dose, evaluated in a trial in which delamanid was administered as 100 mg BID for 8 weeks plus 200 mg QD for 18 weeks, was 89.3% based on the mouse PDT and >90% on the other PDTs. QTcF (QTc interval corrected for heart rate by Fridericia's formula) prolongation was approximately 50% lower for the 200 mg QD dose than the 100 mg BID dose. In conclusion, while CFRs of 100 mg BID and 200 mg QD delamanid were close to or above 90% in patients with MDR-TB, more-convenient once-daily dosing of delamanid is feasible and likely to have less effect on QTcF prolongation.

sncRNA-1 Is a Small Noncoding RNA Produced by Mycobacterium tuberculosis in Infected Cells That Positively Regulates Genes Coupled to Oleic Acid Biosynthesis.

Nearly one third of the world's population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different Mtb encoded proteins in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. We performed RNA sequencing and identified a subset of Mtb H37Rv-encoded small RNAs (<30 nts in length) that were produced in infected macrophages. Designated as smaller noncoding RNAs (sncRNAs), three of these predominated the read counts. Each of the three, sncRNA-1, sncRNA-6, and sncRNA-8 had surrounding sequences with predicted stable secondary RNA stem loops. Site-directed mutagenesis of the precursor sequences suggest the existence of a hairpin loop dependent RNA processing mechanism. A functional assessment of sncRNA-1 suggested that it positively regulated two mycobacterial transcripts involved in oleic acid biosynthesis. Complementary loss- and gain- of-function approaches revealed that sncRNA-1 positively supports Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Overall, the findings reveal that Mtb produces sncRNAs in infected cells, with sncRNA-1 modulating mycobacterial gene expression including genes coupled to oleic acid biogenesis.

Comparison of a Novel Regimen of Rifapentine, Tedizolid, and Minocycline with Standard Regimens for Treatment of Pulmonary Mycobacterium kansasii.

The combination of isoniazid, rifampin, and ethambutol is recommended by the American Thoracic Society (ATS) for treatment of pulmonary Mycobacterium kansasii, while the British Thoracic Society (BTS) recommends clarithromycin, rifampin and ethambutol. Unfortunately, therapy duration for both regimens lasts for years. In this study, we administered tedizolid, minocycline, clarithromycin, and rifapentine as monotherapy as well as novel combinations in the intracellular hollow-fiber model system of M. kansasii (HFS-Mkn) in a 28-day study. The ATS and BTS regimens were used as comparators. Repetitive sampling was used to validate the intended intrapulmonary pharmacokinetics of each drug and to monitor changes in M. kansasii burden. As monotherapy, tedizolid at an observed area under the concentration-time curve from 0 to 24 h (AUC0-24)/MIC of 5.85 and minocycline at an AUC0-24/MIC of 5.77 failed to kill the bacteria below day 0 (stasis), clarithromycin at an AUC0-24/MIC of 2.4 held the bacterial burden at stasis, but rifapentine at an AUC0-24/MIC of 140 killed 2 log10 CFU/ml below stasis. The BTS regimen kill slope was -0.083 ± 0.035 CFU/ml/day, which was significantly superior to the ATS regimen slope of -0.038 ± 0.038 CFU/ml/day. The rifapentine-tedizolid-minocycline combination kill slope was -0.119 ± 0.031 CFU/ml/day, superior to that of the ATS regimen and comparable to that of the BTS regimen. In conclusion, the BTS regimen and the novel rifapentine-tedizolid-minocycline regimen showed better kill of intracellular bacteria in the HFS-Mkn However, the efficacy of the new combination regimen remains to be tested in clinical settings.

Effect of specimen processing, growth supplement, and different metabolic population on Mycobacterium tuberculosis laboratory diagnosis.

INTRODUCTION: Sputum specimen decontamination steps are essential due to the presence of other saprophytic and infectious organisms. However, they negatively affect the mycobacterial recovery. In addition, little is known about the Mycobacterium tuberculosis killing efficacy of the PANTA (polymyxin-B, amphotericin-B, nalidixic acid, trimethoprim, azilocillin) antibiotics. Moreover, M. tuberculosis can be present in more than one metabolic population, but the effect of different growth characteristics on the mycobacterial growth indicator tube (MGIT) based time-to-positive (TTP) is not well studied. METHODS: We performed-(1) experiments using the solid agar and MGIT method to determine the effect of the NALC-NaOH decontamination method, (2) concentration-response studies with each individual antibiotic in the PANTA, and (3) the effect of the M. tuberculosis metabolic population on the TTP. TTP was recorded using the Epicenter software and exponential growth equation was used to calculate the doubling time of the bacteria, whereas, CFU/mL was analyzed using the Inhibitory Sigmoid Emax model for each antibiotic. RESULTS: Decontamination resulted in 4.36+0.13 log10 CFU/mL difference in cultures treated with NALC-NaOH versus no decontamination process and the limit of detection decreased from 1.47 log10 CFU/mL to the 0.42 log10 CFU/mL following NALC-NaOH treatment. PANTA at currently used antibiotic concentrations, did not had negative effect on mycobacterial recovery. Exponential growth model estimated doubling time for the log-phase growth M. tuberculosis as 2.04 days, for the semi-dormant bacilli as 2.80 days, and 6.37 days for the anaerobic cultures. CONCLUSION: Specimen decontamination method negatively affect the laboratory diagnosis of M. tuberculosis, polymyxin-B and nalidixic acid have anti-tuberculosis efficacy at high concentrations, and the doubling time of different metabolic population should be considered when deciding the time-in-protocol for the MGIT system.

d-Cycloserine Pharmacokinetics/Pharmacodynamics, Susceptibility, and Dosing Implications in Multidrug-resistant Tuberculosis: A Faustian Deal.

Background: d-cycloserine is used to treat multidrug-resistant tuberculosis. Its efficacy, contribution in combination therapy, and best clinical dose are unclear, also data on the d-cycloserine minimum inhibitory concentration (MIC) distributions is scant. Methods: We performed a systematic search to identify pharmacokinetic and pharmacodynamic studies performed with d-cycloserine. We then performed a combined exposure-effect and dose fractionation study of d-cycloserine in the hollow fiber system model of tuberculosis (HFS-TB). In parallel, we identified d-cycloserine MICs in 415 clinical Mycobacterium tuberculosis (Mtb) isolates from patients. We utilized these results, including intracavitary concentrations, to identify the clinical dose that would be able to achieve or exceed target exposures in 10000 patients using Monte Carlo experiments (MCEs). Results: There were no published d-cycloserine pharmacokinetics/pharmacodynamics studies identified. Therefore, we performed new HFS-TB experiments. Cyloserine killed 6.3 log10 colony-forming units (CFU)/mL extracellular bacilli over 28 days. Efficacy was driven by the percentage of time concentration persisted above MIC (%TMIC), with 1.0 log10 CFU/mL kill achieved by %TMIC = 30% (target exposure). The tentative epidemiological cutoff value with the Sensititre MYCOTB assay was 64 mg/L. In MCEs, 750 mg twice daily achieved target exposure in lung cavities of 92% of patients whereas 500 mg twice daily achieved target exposure in 85% of patients with meningitis. The proposed MCE-derived clinical susceptibility breakpoint at the proposed doses was 64 mg/L. Conclusions: Cycloserine is cidal against Mtb. The susceptibility breakpoint is 64 mg/L. However, the doses likely to achieve the cidality in patients are high, and could be neurotoxic.

Ceftazidime-avibactam has potent sterilizing activity against highly drug-resistant tuberculosis.

There are currently many patients with multidrug-resistant and extensively drug-resistant tuberculosis. Ongoing transmission of the highly drug-resistant strains and high mortality despite treatment remain problematic. The current strategy of drug discovery and development takes up to a decade to bring a new drug to clinical use. We embarked on a strategy to screen all antibiotics in current use and examined them for use in tuberculosis. We found that ceftazidime-avibactam, which is already used in the clinic for multidrug-resistant Gram-negative bacillary infections, markedly killed rapidly growing, intracellular, and semidormant Mycobacterium tuberculosis in the hollow fiber system model. Moreover, multidrug-resistant and extensively drug-resistant clinical isolates demonstrated good ceftazidime-avibactam susceptibility profiles and were inhibited by clinically achievable concentrations. Resistance arose because of mutations in the transpeptidase domain of the penicillin-binding protein PonA1, suggesting that the drug kills M. tuberculosis bacilli via interference with cell wall remodeling. We identified concentrations (exposure targets) for optimal effect in tuberculosis, which we used with susceptibility results in computer-aided clinical trial simulations to identify doses for immediate clinical use as salvage therapy for adults and young children. Moreover, this work provides a roadmap for efficient and timely evaluation of antibiotics and optimization of clinically relevant dosing regimens.

The discovery of ceftazidime/avibactam as an anti-Mycobacterium avium agent.

OBJECTIVES: To determine if ceftaroline and ceftazidime combined with avibactam are efficacious against pulmonary Mycobacterium avium complex (MAC) disease. METHODS: First, we performed a concentration-effect study of ceftaroline and ceftaroline/avibactam against extracellular MAC in test tubes. Given the difficulty of obtaining avibactam at the time of experimentation, we used a single concentration of commercial ceftazidime/avibactam, and two sets of non-treated controls, one with ceftazidime/avibactam and the other without. After finding antimicrobial activity with the ceftazidime/avibactam 'control', we performed ceftazidime/avibactam dose-effect studies in test tubes against extracellular MAC and in 24-well plates against intracellular MAC. We then performed a ceftazidime/avibactam exposure-effect and dose-fractionation studies in the hollow-fibre system model of intracellular pulmonary MAC (HFS-MAC). In each experiment, we repetitively sampled each HFS-MAC at specified times to validate ceftazidime/avibactam pharmacokinetics and to quantify bacterial burden. RESULTS: Ceftaroline killed extracellular MAC with maximal microbial kill (Emax) of 4.87 ±âŸ0.26 log10 cfu/mL. However, the ceftazidime/avibactam 'control' also killed MAC compared with the non-treated control. Ceftazidime/avibactam Emax was 3.8 log10 cfu/mL against extracellular bacilli and 3.6 log10 cfu/mL against intracellular MAC. In the HFS-MAC, ceftazidime/avibactam achieved a half-life of 2.5-3.3 h and killed MAC 0.61-2.40 log10 cfu/mL below the starting bacterial burden. The ceftazidime/avibactam efficacy was linked to the proportion of the dosing interval for which the concentration persists above the MIC (fT>MIC), with optimal efficacy at free-drug fT>MIC of 52% (r2 = 0.95). CONCLUSIONS: Ceftazidime/avibactam effectively kills MAC at exposures easily achieved in the lung by clinical doses. Efficacy was higher than with clinically achievable doses of azithromycin and ethambutol.

Neonatal Septicemia in Nepal: Early-Onset versus Late-Onset.

Introduction. Neonatal septicemia is defined as infection in the first 28 days of life. Early-onset neonatal septicemia and late-onset neonatal septicemia are defined as illnesses appearing from birth to three days and from four to twenty-eight days postnatally, respectively. Methods. In this cross-sectional study, blood samples from the suspected infants were collected and processed in the bacteriology laboratory. The growth was identified by standard microbiological protocol and the antibiotic sensitivity testing was carried out by modified Kirby-Bauer disk diffusion method. Results. Among total suspected cases, the septicemia was confirmed in 116 (12.6%) neonates. Early-onset septicemia (EOS) was observed in 82 infants and late-onset septicemia (LOS) in 34 infants. Coagulase-negative staphylococcus (CoNS) (46.6%) was the predominant Gram-positive organism isolated from EOS as well as from LOS cases followed by Staphylococcus aureus (14.6%). Acinetobacter species (9.5%) was the predominant Gram-negative organism followed by Klebsiella pneumoniae (7.7%). Conclusions. The result of our study reveals that the CoNS, Staphylococcus aureus, Acinetobacter spp., and Klebsiella pneumoniae are the most common etiological agents of neonatal septicemia. In particular, since rate of CoNS causing sepsis is alarming, prompting concern to curb the excess burden of CoNS infection is necessary.

Community acquired multi-drug resistant clinical isolates of Escherichia coli in a tertiary care center of Nepal.

BACKGROUND: Multi-drug resistance (MDR) in Gram-negative organisms is an alarming problem in the world. MDR and extensively-drug resistance (XDR) is in increasing trend due to the production of different types of beta (ß)-lactamases. Thus the aim of this study was to document the incidence of MDR and XDR in clinical isolates of Escherichia coli and also to find out the enzymatic mechanisms of ß-lactam antibiotics resistance. METHODS: Two hundred clinical isolates of Escherichia coli (E. coli) identified by standard laboratory methods were studied. Antibiotic susceptibility profile was performed for all the isolates and the suspected isolates were phenotypically tested for the production of extended spectrum ß-lactamase (ESBL), metallo ß-lactamase (MBL) and AmpC ß-lactamase (AmpC) by recommended methods. RESULTS: Around three-fourth (78%) of the total isolates were multi-drug resistant. ESBL, MBL and AmpC production was found in 24%, 15% and 9% of isolates respectively. Amikacin, chloramphenicol and colistin were found to be the most effective antibiotics. CONCLUSIONS: High percentage of MDR was observed. ß-lactamase mediated resistance was also high. Thus, regular surveillance of drug resistance due to ß-lactamases production and infection control policy are of utmost importance to minimize the spread of resistant strains.

Threat of drug resistant Staphylococcus aureus to health in Nepal.

BACKGROUND: Staphylococcus aureus is the most commonly isolated organism from the different clinical samples in hospital. The emergence and dissemination of methicillin resistant Staphylococcus aureus (MRSA) and growing resistance to non-beta-lactam antibiotics is making treatment of infections due to this organism increasingly difficult. METHODS: This study was conducted to determine the frequency of Staphylococcus aureus isolated from different clinical samples, rates of MRSA and full antibiotic susceptibility profiles. Clinical samples were cultured and Staphylococcus aureus was identified using standard microbiological methods recommended by the American Society for Microbiology (ASM). Methicillin resistance was confirmed using cefoxitin and oxacillin disks. Inducible clindamycin resistance was identified using D-zone test. RESULTS: From the processed samples, 306 isolates of Staphylococcus aureus were recovered. All the isolates were susceptible to vancomycin and teicoplanin. Methicillin resistance was observed in 43.1% of isolates while inducible clindamycin resistance in 12.4% of the isolates. CONCLUSIONS: The results of our study reveals that rates of resistance to commonly prescribed antibiotics in Staphylococcus aureus clinical isolates is high. In particular, rate of methicillin resistance is alarming, prompting concern on the rational use of antibiotics and vigilant laboratory-based surveillance of resistance rates in Nepal.

Childhood septicemia in Nepal: documenting the bacterial etiology and its susceptibility to antibiotics.

Introduction. Children are among the most vulnerable population groups to contract illnesses. The varying microbiological pattern of septicemia warrants the need for an ongoing review of the causative organisms and their antimicrobial susceptibility pattern. Therefore, the objective of this study was to document the bacterial etiology of childhood septicemia and its antibiotic susceptibility profile. Methods. Cross-sectional type of study in 1630 suspected patients was conducted at CMCTH from January 2012 to December 2013. Blood samples were collected aseptically for culture. The organisms grown were identified by standard microbiological methods recommended by American Society for Microbiology (ASM) and subjected to antibiotic susceptibility testing by modified Kirby-Bauer disk diffusion method. Methicillin resistance was confirmed using cefoxitin and oxacillin disks methods. Results. Septicemia was detected in 172 (10.6%) cases. Among Gram-positive organisms, coagulase negative staphylococci (CoNS) were leading pathogen and Acinetobacter spp. were leading pathogen among Gram-negative isolates. Vancomycin, teicoplanin, and clindamycin were the most effective antibiotics against Gram-positive isolates while amikacin was effective against Gram-positive as well as Gram-negative isolates. Methicillin resistance was detected in 44.4% of Staphylococcus aureus. Conclusions. This study has highlighted the burden of bacterial etiology for septicemia among children in a tertiary care center of central Nepal.

Stimulation of unprimed macrophages with immune complexes triggers a low output of nitric oxide by calcium-dependent neuronal nitric-oxide synthase.

Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca(2+)) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca(2+) flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca(2+)-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca(2+) flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca(2+)-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.

Human polyomavirus JC (JCV) infection of human B lymphocytes: a possible mechanism for JCV transmigration across the blood-brain barrier.

It has been suggested that JC virus (JCV) might travel to the central nervous system in infected B cells. Moreover, recent data suggest the presence of JCV in bone marrow plasma cells. However, the evidence for infection and replication of JCV in B cells is unclear. To address this question, we infected Epstein-Barr virus-transformed B cells with JCV and found that the viral genome decreased >1000-fold from days 0 to 20 after infection, which concurred with the absence of viral early and late messenger RNA transcripts and proteins. However, immunofluorescent images of B cells infected with fluorescein isothiocyanate-conjugated JCV demonstrated that JCV enters the B cells, and DNase protection assay confirmed the presence of intact JCV virions inside the B cells. Moreover, JCV-infected B cells were able to transmit infection to naive glial cells. These data confirm that JCV nonproductively infects B cells and possibly uses them as a vehicle for transmigration across the blood-brain barrier.

West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier.

Neurological complications such as inflammation, failure of the blood-brain barrier (BBB), and neuronal death contribute to the mortality and morbidity associated with WNV-induced meningitis. Compromised BBB indicates the ability of the virus to gain entry into the CNS via the BBB, however, the underlying mechanisms, and the specific cell types associated with WNV-CNS trafficking are not well understood. Brain microvascular endothelial cells, the main component of the BBB, represent a barrier to virus dissemination into the CNS and could play key role in WNV spread via hematogenous route. To investigate WNV entry into the CNS, we infected primary human brain microvascular endothelial (HBMVE) cells with the neurovirulent strain of WNV (NY99) and examined WNV replication kinetics together with the changes in the expressions of key tight junction proteins (TJP) and cell adhesion molecules (CAM). WNV infection of HBMVE cells was productive as analyzed by plaque assay and qRT-PCR, and did not induce cytopathic effect. Increased mRNA and protein expressions of TJP (claudin-1) and CAM (vascular cell adhesion molecule and E-selectin) were observed at days 2 and 3 after infection, respectively, which coincided with the peak in WNV replication. Further, using an in vitro BBB model comprised of HBMVE cells, we demonstrate that cell-free WNV can cross the BBB, without compromising the BBB integrity. These data suggest that infection of HBMVE cells can facilitate entry of cell-free virus into the CNS without disturbing the BBB, and increased CAM may assist in the trafficking of WNV-infected immune cells into the CNS, via 'Trojan horse' mechanism, thereby contributing to WNV dissemination in the CNS and associated pathology.

Serotonin receptor 2A blocker (risperidone) has no effect on human polyomavirus JC infection of primary human fetal glial cells.

A recent report demonstrated that JC virus (JCV) employs serotonin receptor 2A (5HT(2A)R) to infect the glial cells. To assess the ability of a potent 5HT(2A)R blocker, risperidone, to inhibit JCV infection, the authors treated primary human fetal glial (PHFG) cells in vitro with risperidone for 24 h and inoculated with JCV(Mad1). There was no significant difference in JCV genome copies or mRNA transcripts and protein expression in treatment-naive and risperidone-treated PHFG cells. These data indicate that risperidone does not inhibit JCV(Mad1) attachment, internalisation, and replication in PHFG cells, and 5HT(2A)R blockers may not be effective in treating progressive multifocal leukoencephalopathy (PML).