Rapid antimicrobial susceptibility profiling using impedance spectroscopy.

The present antibiotic susceptibility testing (AST) techniques based on bacterial culture, gene amplification and mass spectrometry are highly time consuming, labour intensive or expensive. Impedance spectroscopy is an emerging tool for rapid bacterial analysis as it is label-free, real-time, affordable and high-throughput. The over-reliance of this technique on complex chip designs and cell enrichment strategies has, however, slowed its foray into clinical AST. We demonstrate a label-free approach in which a low conductivity zwitterionic buffer is used for boosting impedance sensitivity in simple interdigitated electrodes (IDEs) allowing rapid AST in just 20 min without any liquid flow, biofunctionalization or cell enrichment steps. The detection principle relies on measuring changes in solution resistance due to antibiotic-induced bacterial cell death or growth. While the death-based approach is faster (20 min), it's restricted to surface-acting bactericidal antibiotics. The cell growth approach is longer (60-80 min) but more versatile as it applies to all drug types. Results for antibiotic sensitivity analysis and minimum inhibitory concentration (MIC) determination are illustrated for Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus against a wide class of antibiotics (penicillins, cephalosporins, polymyxins, carbapenems etc.).

Th2 immune response by the iron-regulated protein HupB of Mycobacterium tuberculosis.

BACKGROUND: HupB is an iron-regulated protein essential for the growth of Mycobacterium tuberculosis inside macrophages. To investigate if HupB induced a dominant Th2 type immune response, we studied the effect of rHupB on PBMCs from TB patients and by infecting mouse macrophages with wild type and hupB KO mutants. METHODS: PBMCs from pulmonary TB (n = 60), extra pulmonary TB (n = 23) and healthy controls (n = 30) were stimulated with purified HupB and the cytokines secreted were assayed. The sera were screened for anti-HupB antibodies by ELISA. Mouse macrophages cell line (RAW 264.7) was infected with wild type, hupB KO and hupB-complemented strains of M. tuberculosis grown in high and low iron medium and the expression of cytokines was assayed by qRT-PCR. RESULTS: Murine macrophages infected with the hupB KO strain produced low levels of the pro-inflammatory cytokines IFN-γ, TNF-α, IL-1, and IL-18 and high levels of IL-10. HupB induced IL-6 and IL-10 production in PBMCs of TB patients and down-regulated IFN-γ and TNF-α production. The influence of HupB was remarkable in the EPTB group. CONCLUSION: HupB shifted the immune response to the Th2 type. Low IFN-γ and elevated IL-10 in EPTB patients is noteworthy.

Direct Detection and Discrimination of Carbapenemases of Acinetobacter baumannii from Uncultured Tracheal Aspirates.

Carbapenemases play important roles in conferring resistance to beta-lactam antibiotics, including the carbapenems. Detection of carbapenemase activity helps to understand the possible mechanism(s) of carbapenem resistance. Identification of carbapenemases is currently being done by various phenotypic methods and molecular methods. However, innovative biochemical and spectrophotometric methods are desirable as they will be easy to perform, affordable, and rapid. A novel chromogenic method called Carba NP test was introduced recently to screen for carbapenemases in clinical isolates of gram-negative pathogens. We adopted this assay (1) to detect the total carbapenemase activity, (2) to discriminate Class A, B, and D carbapenemases with inhibitors, (3) to compare with carbapenemase genotype, and (4) for direct differential diagnosis of carbapenemases in uncultured clinical sample such as tracheal aspirate. The study included 132 purulent tracheal aspirates. All samples were processed and screened by a protocol optimized in our laboratory, which showed good sensitivity and correlation with genotyping and conventional phenotyping. Our protocol not only offers the fastest way to identify the pathogen but also its carbapenemase profile, directly from uncultured clinical samples in less than 4 hr. Our protocol is currently being validated on other types of clinical specimens in our laboratory.

ε-Polylysine Nanoconjugates: Value-Added Antimicrobials for Drug-Resistant Bacteria.

Antimicrobial resistance poses a serious threat to human health and is evidently not restricted to any one part of the globe. Over the last few decades, no new antibiotics have been discovered, and many antibiotics currently available are failing against several critical pathogenic strains due to emerging drug resistance. We have designed a strategy to combat deadly drug-resistant bacteria by using nanocargos that consist of gold nanoparticles (AuNPs) conjugated to ε-polylysine (PLL) and octadecanethiol (C18) either alone or in combination. These nanocargos when tested against reference strains of carbapenem-resistant Acinetobacter baumannii (CRAB) and methicillin-resistant Staphylococcus aureus (MRSA) showed 15-20-fold higher antibacterial activity compared to free PLL. The minimum inhibitory concentration (MIC) of the nanoconjugates was found to lie between 8 and 15 µg/mL for both these bacteria, and they were also found to be nonhemolytic and nontoxic to mammalian cells. The mechanistic evaluation of antibacterial action showed alternate pathways of uptake for free PLL and the nanoconjugates. Further, the nanocargos were successfully used and found to be superior to free PLL in preventing biofilm formation in MRSA and CRAB. The PLL nanoconjugates may find applications in prevention of bacterial biofilm formation on surfaces such as surgical instruments and indwelling devices like stents, catheters, cannulas, orthopedic implants, and pacemakers.

A Flowthrough Assay for Rapid Bedside Stratification of Bloodstream Bacterial Infection in Critically Ill Patients: a Pilot Study.

Bacterial infections affect more than 2 million people annually. Of these, systemic infections caused by bacteria in critically ill patients may lead to life-threatening conditions such as sepsis. We have developed a point-of-care (POC) device called Septiflo that can detect and stratify the Gram status of bloodstream bacterial infections in less than 10 min from a drop of human plasma. It works on the principle of identifying pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS) and lipoteichoic acid (LTA) that are released into the bloodstream at the onset of Gram-negative and Gram-positive bacterial infections, respectively. The biomarkers are captured on a membrane without a receptor, and the Gram status specificity is conferred by the ligands attached to gold nanoparticles (AuNPs) used as signal amplification probes. The ultrasensitive colorimetric results are read by eye down to a 100-fg/ml detection limit without an instrument. No cross-interference between the PAMPs is seen during Gram stratification. Septiflo results also display better performance than commercial enzyme-linked immunosorbent assays (ELISAs). Tests performed on 60 clinical samples from patients showed a correlation accuracy of 70% against procalcitonin (PCT), an accepted surrogate biomarker for sepsis. A direct comparison with eubacterial PCR yielded up to 94% accuracy in 31 patients at a chosen cutoff level for LPS and LTA and area under the curve (AUC) values of 0.927 and 0.885, respectively, though blood culture was negative for most samples. The high sensitivity, low cost, and simple bedside utility of the assay may aid in better sepsis management apparently at the presymptomatic stage, lowering empirical therapy, medical costs, antimicrobial resistance, and mortality.

In vitro flow-through assay for rapid detection of endotoxin in human sera: A proof-of-concept.

An increase in endotoxin concentration in the bloodstream can trigger activation of innate immune response leading to septic shock. There is currently no method available for rapid endotoxin detection at a patient's bedside. We demonstrate a simple, portable and cost-effective strategy to measure endotoxin levels in human serum within 5min using a flow-through assay. A drop of serum containing LPS was spotted on an endotoxin-affinity membrane placed over high-wicking absorbent pads. Subsequent addition of polymyxin B sulfate drug-conjugated gold nanoparticles allowed concentration-dependent visualization of spots by the naked eye in the clinically-relevant range of 10pg/mL to 10ng/mL. The results were quantified using a concentration-calibrated color chart and the assay performance was tested with archival plasma samples of 18 known septicemia patients. The results showed a reasonably good correlation with the patients' hematological data. This proof-of-concept study puts forth an interesting alternative for early septicemia diagnosis in future.

Nanotheranostic approaches for management of bloodstream bacterial infections.

Bloodstream bacterial infections are a serious threat to global public health and economy. The recent figures released by National Center for Health Statistics indicate that more than a million Americans get affected by it each year and the sepsis mortality alone is about 28%-50% Hall et al. (2011).1 Robust and affordable point-of-care medical technologies are, therefore, urgently needed for rapid decision-making to initiate appropriate line of treatment. Current techniques based on blood culture and serology do not have quick turnaround times or adequate sensitivities for early intervention. Moreover, antimicrobial resistance poses a great challenge in the fight towards effective bacterial infection management. Nanotheranostics is emerging as a novel strategy combining solutions for rapid diagnosis and treatment in a more personalized way. This review highlights the recent advances made in theranosis of bloodstream bacterial infections using different classes of nanomaterials and bioreceptors, and discusses present challenges and future way forward.

Continuous evaluation of changes in the serum proteome from early to late stages of sepsis caused by Klebsiella pneumoniae.

Serum protein profiles of patients with bacterial sepsis from the day of diagnosis until recovery/mortality were compared from early to late stages in response to severe sepsis using two dimensional electrophoresis. The proteins exhibiting changes during the course of sepsis (20­28 day mortality) were selected and identified by matrix­assisted laser desorption ionization­time of flight­tandem mass spectrometry. Among the proteins identified, haptoglobin (Hp), transthyretin (TTR), orosomucoid 1/α1 acid glycoprotein (ORM1), α1 antitrypsin (A1AT), serum amyloid A (SAA) and S100A9 exhibited differential expression patterns between survivors (S; n=6) and non­survivors (NS; n=6), particularly during the early stages of sepsis. Expression factors (EFs), taken as the ratio between the NS and S during early stages, showed ratios of Hp, 0.39 (P≤0.012); TTR, 3.96 (P≤0.03); ORM1, 0.69 (P≤0.79); A1AT, 0.92 (P≤0.87) and SAA, 0.69 (P≤0.01). S100A9, an acute phase protein, exhibited an EF ratio of 1.68 (P≤0.004) during the end stages of sepsis. A delayed rise in levels was observed in Hp, A1AT, ORM1, S100A9 and SAA, whereas TTR levels increased during the early stages of sepsis in NS. Analysis of inflammatory responses in the early stages of sepsis revealed increased mRNA expression in leukocytes of interleukin (IL)­6 (EF, 2.50), IL­10 (EF, 1.70) and prepronociceptin (EF, 1.6), which is a precursor for nociceptin in NS compared with S, and higher Toll­like receptor­4 (EF, 0.30) levels in S compared with NS. Therefore, a weaker acute phase response in the early stages of sepsis in NS, combined with an inefficient inflammatory response, may contribute to sepsis mortality.

Nanoparticle-Drug Bioconjugate as Dual Functional Affinity Ligand for Rapid Point-of-Care Detection of Endotoxin in Water and Serum.

Endotoxin or lipopolysaccharide (LPS) is a major constituent of the Gram-negative bacterial cell wall that causes a life-threatening disorder called septicemia resulting from the unregulated activation of the innate immune system. We demonstrate a simple and robust drug-assisted dot blot bioassay for endotoxin detection that can be used right by the critically ill patients' bedside. Target LPS molecules are trapped from serum or water on glass substrates via long-chain alkyls and tagged with reporter gold nanoparticles (NPs) preconjugated to an antibiotic drug called polymyxin B sulfate (PMB). A post-silver-enhancement step enables signal visibility to the bare eye over a wide and clinically relevant concentration range of 50 fg/mL-50 ng/mL, allowing effortless diagnosis of sepsis at various stages, from early sepsis to septic shock.

A single centre prospective study of liver function tests in post liver transplant patients.

Liver transplantation means surgical replacement of a diseased liver with a healthy liver. The survival rate used to be 30 % after 1 year and LTx was considered to be the last procedure when all medical or surgical intervention failed. Advances in donor organ preservation, surgical techniques, patient selection, immunosuppressive regimens and treatments for opportunistic infections all have contributed to substantially improve the survival rates. Despite substantial technological, medical and surgical advances, liver transplantation remains a complex procedure that is accompanied by significant morbidity and mortality. The post-operative outcome of each patient varies greatly depending on the patient's pre- operative state, quality of the donated organ and the complexity of the surgery. Complications occur both immediately post transplant and in the long term. Most of the problems can be satisfactorily assessed with a panel of routine LFTs results of which are generated quickly, cheaply on the analyzer which operates 24 h. Liver Function Test identifies the presence of problem but not problem itself. Abnormal results can be meaningful only when used with clinical data, radiological findings. The study includes 75 post LTx patients in three groups adults (non ACR), Pediatrics and ACR. All recipients were on immunosuppressive therapy (tacrolimus, mycophenolate and methylprednisolone), antiviral (ganciclovir), antiprotozoal, antibacterial and antifungal (fluconazole). 5 mL of blood was drawn in plain vacutainer from the post LTx patients every day for 15 days and LFT and GGT was done. Routinely performed liver function tests correlates well with clinical complications involving liver in the transplant patients. Instead of daily testing, may be alternate day analysis of LFT should be sufficient for effective monitoring of patients. The total protein and albumin and the transaminases offer little help in monitoring LFT post LTx. The elevated levels of serum GGT and ALP may be related to chronic immune damage to the transplanted liver. Serum GGT and ALP can be used as early markers for diagnosing biliary complications and can be used to asses adequacy of endoscopic treatment in the group of patients presenting early. Thus, most of the problems can be satisfactorily assed with a panel of routine LFTs generated quickly, cheaply on analyzer which operates 24 h each day. However, it must be emphasized that LFTs may identify the presence of problems but not the problem itself and the abnormal results are meaningful only when correlated with other clinical information.

Pharmacokinetics of tacrolimus in adult renal transplant recipients.

BACKGROUND: The success of an immunosuppressive drug therapy depends on the extent of exposure to the drugs (the blood levels and duration), which is measured as the area under the curve (AUC). Tacrolimus shows considerable variability in its pharmacokinetics, with poor correlation between the tacrolimus trough level and systemic exposure, as measured by the AUC of concentration time. Monitoring trough levels helps not only in reducing nephrotoxiicity but also in reducing the chances of acute rejection; although there is no international consensus, the trough concentration is used to determine dosing and the AUC for calculating the exposure of the patient to the drug. The major objective of this study was to find the best sampling time for an abbreviated AUC0-6 (area under the concentration time curve) to predict the total body exposure to tacrolimus in adult renal transplantation recipients. METHODS: The study involved retrospective analysis of 14 renal transplant patients (2 female and 12 male) that were on triple immunosuppressive therapy, methyl prednisolone, mycophenolate mofetil and tacrolimus. To determine trough concentrations, blood samples were collected before administration of tacrolimus (0 h) and at fixed time points of 2 h, 4 h and 6 h after administration of oral tacrolimus and analyzed in duplicate by microparticle enzyme immunoassay. AUC0-6 was determined using the linear trapezoidal rule. The association between the blood concentration and AUC6 were evaluated by the Pearson correlation coefficient. All statistical analyses were performed using the SPSS software (IBM Corp., NY, USA) program. RESULTS: Trough levels were fairly consistent at 7.9-18 ng·h/mL in all the patients included in this study, and this did not show variation with age or sex. The AUC0-6 was higher [202-290 ng/mL at 3-8 mg bis-daily (b.d.) dosage] in patients who received kidneys from cadavers compared to recipients from live donors (60.5-171 ng/mL at 3-8 mg b.d. dosage), but the clinical significance of this is not known. The highest AUC0-6 was 246 ng/mL, observed at 4.5 mg b.d. dosage. Dosages higher than 2 mg b.d. did not result in a noticeable increase in AUC0-6. Peak blood levels of tacrolimus were obtained 4 h after administration. CONCLUSIONS: Trough level determination and a C2, C4 two-point limited sampling strategy may be useful to plan the dosing strategy and estimate the exposure of renal transplant patients to tacrolimus.