Use Of Isothermal Microcalorimetry Research Articles

6. Utility of next generation sequencing and isothermal microcalorimetry for identification of bacteria in presumed aseptic thoracolumbar revision spine surgery

<h3>BACKGROUND CONTEXT</h3> Recent studies have been able to detect the subclinical presence of bacteria in 10%-40% of patients undergoing revision spinal instrumentation procedures, suggesting that clinically silent bacteria may be responsible for presumed aseptic revisions. <h3>PURPOSE</h3> The purpose of this study is to determine the rate of bacterial presence in revision thoracolumbar spinal instrumentation surgery due to implant failure. In patients without clinical signs or symptoms of infection, we will utilize next generation sequencing (NGS) technology as well as cultures and isothermal microcalorimetry (IMC) to determine the rate of subclinical infection. <h3>STUDY DESIGN/SETTING</h3> A prospective pilot case-series at a single academic institution between September 2020 and September 2021. <h3>PATIENT SAMPLE</h3> Patients undergoing consecutive thoracolumbar revision spine surgery resulting from implant failure. <h3>OUTCOME MEASURES</h3> The main outcome measure was bacterial presence. Routine patient and clinical information including age, sex, body mass index, preoperative indication, and additional clinical/operative information were also identified. Lab values included ESR, CRP and procalcitonin. COVID tests results were documented. <h3>METHODS</h3> Patients undergoing thoracolumbar revision spine surgery due to implant failure by a single surgeon at a single institution were prospectively identified. Tissue and blood samples were taken intraoperatively and transported to a lab for analysis using IMC and NGS. <h3>RESULTS</h3> In the pilot study, 22 consecutive patients were included based on prespecified criteria. The average (SD) age of the patients was 65 (14) with 25% female sex. There were 82 total tissue samples obtained (mean 3.7 samples per patient), with a concordance of 93% between cultures and IMC, 85% between cultures and NGS, and 88% between IMC and NGS. The prevalence of occult surgical site infection was detected in 4/22 (18%) cases by NGS, 2/22 (9%) cases by IMC, and 0/22 (0%) by standard cultures. Two out of two (100%) infections detected by IMC and 2/4 (50%) of the NGS infections had grossly loose/broken pedicle screws without clear etiology for breaking, suggesting these are potentially real subclinical infection cases. There was one additional loosening of screws case without pathogen identification by any platform. However, no patients have undergone subsequent surgery for concern for infection to date. For that reason, considering loose/broken screw cases as true subclinical infections IMC had 67% sensitivity, 100% specificity, 100% PPV, and 95% NPV, while NGS was 67%, 90%, 50%, 94% respectively, and cultures were 0%, 100%, 0%, 87% respectively. Pathogens identified include Fusarium equiseti (fungi), Acinetobacter spp., Brevundimonas spp., Lecythophora spp. (fungi), Enterobacter cloacae, Escherichia coli, Enterococcus faecalis, and Staphylococcus haemolyticus. <h3>CONCLUSIONS</h3> The detection of infection in spinal implants is challenging. The use of IMC and NGS identified two occult cases of infection, while NGS also falsely identified two additional cases of infection in our pilot case series of thoracolumbar revision spine surgery, while one possible subclinical infection had no pathogens identified on any platform. Research using IMC and NGS to detect subclinical infection in revision spine surgery requires further investigation but indicates subclinical infection may be an explanation for some instances of revision surgery, particularly those involving loose or broken pedicle screws. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Detection and Drug Susceptibility Testing of Neisseria gonorrhoeae Using Isothermal Microcalorimetry.

Background: Gonorrhea is a frequently encountered sexually transmitted disease that results in urethritis and can further lead to pelvic inflammatory disease, infertility, and possibly disseminated gonococcal infections. Thus, it must be diagnosed promptly and accurately. In addition, drug susceptibility testing should be performed rapidly as well. Unfortunately, Neisseria gonorrhoea is a fastidious microorganism that is difficult to grow and requires culturing in an opaque medium. Methods: Here, we used isothermal microcalorimetry (IMC) to monitor the growth and the antimicrobial susceptibility of N. gonorrhoea. Results: Using IMC, concentrations of N. gonorrhoea between 2000 and 1 CFU·mL−1 were detected within 12 to 33 h. In addition, drug susceptibility could be monitored easily. Conclusions: The use of isothermal microcalorimetry provides an interesting and useful tool to detect and characterize fastidious microbes such as N. gonorrhoea that require media incompatible with optical detection conventionally used in many commercial systems.

Isothermal microcalorimetry measures UCP1-mediated thermogenesis in mature brite adipocytes

The activation of thermogenesis in adipose tissue has emerged as an important target for the development of novel anti-obesity therapies. Using multi-well isothermal microcalorimetry, we have demonstrated that mature murine brown and brite adipocytes produce quantifiable heat upon β3-AR stimulation, independently of any anaerobic mechanisms. Additionally, in brite adipocytes lacking UCP1 protein, β3-AR stimulation still induces heat production, albeit to a much lower extent than in their wildtype counterparts, suggesting that UCP1 is an essential component of adrenergic induced thermogenesis in murine brite adipocytes exvivo. Similarly, we could observe an increase in heat production in human-derived adipocytes (hMADS) upon β-AR stimulation. Collectively, these results establish the use of isothermal microcalorimetry as a sensitive and accurate technique for measuring thermogenic responses in intact mature brite adipocytes from murine and human origin.

(Invited) Gaining Insight into Decomposition Reactions within Energy Storage Systems: Use of Operando Isothermal Microcalorimetry

Conversion-type materials for next generation lithium ion systems are of particular interest due to the opportunity for high delivered capacity. However, implementation of conversion materials has been hindered by the phase transformations and large volume changes occurring during cycling. An associated factor is that liquid electrolytes are often not thermodynamically stable at the potentials of the active electrodes while the battery is functioning. This results in the decomposition of electrolyte leading to organic and inorganic interphases formed at the electrode surface. The influence of the interphases can be particularly profound for conversion materials where phase change takes place during (de)lithiation. Notably, the interfaces are critical factors in determining the transport of electrons and ions, the resultant polarization, and associated heat generation. The use of isothermal microcalorimetry (IMC) will be described as an operando tool to determine the heat evolved due to parasitic or decomposition reactions during lithiation. The total heat measured is assessed in light of the contributions from cell polarization, entropic contributions and the parasitic reactions. This study will describe the use of complementary ex-situ and operando characterization techniques to provide insight into decomposition reactions taking place within energy storage systems.

A microcalorimetry study on the oxidation of linoleic acid and the control of rancidity

The oxidative deterioration of unsaturated lipids fatty acids is a great concern for the food industry as it is associated with the development of rancid off-odors and the reduction of food quality. This work describes the potential use of isothermal microcalorimetry to monitor the oxidation of linoleic acid, an important conjugated fatty acid responsible for the development of rancidity. The heat flow signal developed during the oxidation process reflects a multistep mechanism typical of radical chain reactions. From the analysis of the thermograms, it is possible to identify a period of the reaction that occurs with an order equal to 0.5. This period is correlated with the propagation period of the radical chain reaction. In addition, it allows detecting the early appearance of rancidity notes, as confirmed with the analysis of the samples headspace by Proton-Transfer-Reaction Mass Spectrometry (PTR-MS). The proposed approach was finally applied to compare the capacity of natural and synthetic antioxidants to inhibit the oxidation process of linoleic acid and the occurrence of rancidity. The results presented here show the advantages of microcalorimetry to study oxidation reactions and their control.

Development of a flow system for studying biofilm formation on medical devices with microcalorimetry

Isothermal microcalorimetry (IMC) is particularly suited to the study of microbiological samples in complex or heterogeneous environments because it does not require optical clarity of the sample and can detect metabolic activity from as few as 104CFU/mL cells. While the use of IMC for studying planktonic cultures is well established, in the clinical environment bacteria are most likely to be present as biofilms. Biofilm prevention and eradication present a number of challenges to designers and users of medical devices and implants, since bacteria in biofilm colonies are usually more resistant to antimicrobial agents. Analytical tools that facilitate investigation of biofilm formation are therefore extremely useful. While it is possible to study pre-prepared biofilms in closed ampoules, better correlation with in vivo behaviour can be achieved using a system in which the bacterial suspension is flowing. Here, we discuss the potential of flow microcalorimetry for studying biofilms and report the development of a simple flow system that can be housed in a microcalorimeter. The use of the flow system is demonstrated with biofilms of Staphylococcus aureus.

Use of isothermal microcalorimetry to quantify the influence of glucose and antifungals on the growth of Candida albicans in urine

Urinary tract infection (UTI) caused by Candida spp. is an increasing problem in clinical practice. Risk factors include diabetes mellitus, extremes of age, urinary tract abnormalities and indwelling catheters. Here, we determined the applicability of isothermal microcalorimetry (IMC) for the detection and antifungal drug susceptibility testing of Candida albicans in artificial urine. Isothermal microcalorimetry was used to monitor the metabolic heat production rates of C. albicans at 37 °C (μW = μJ s(-1) ). The influence of increasing concentrations of glucose and antifungal drugs on the growth of C. albicans was investigated. The growth rate increased linearly from 0.024 ± 0.010 to 0.203 ± 0.006 h(-1) with increasing concentration of glucose from 20 to 1640 mg l(-1) . The minimum inhibitory concentrations (MIC) against C. albicans were determined at a fixed glucose concentration of 560 mg l(-1) . These MIC were 0.5 μg ml(-1) for amphotericin B, 5 μg ml(-1) for flucytosine, 0.8 μg ml(-1) for fluconazole and 0.5 μg ml(-1) for tioconazole, respectively. IMC is able to detect and quantify growth of C. albicans in artificial urine and to determine the MIC of antifungal drugs against C. albicans. This study demonstrated that IMC can be used for basic research on Candida-UTI and opens promising avenue for the use of IMC as rapid drug resistance screening tool and diagnostic tool. Little is known on the growth of C. albicans in urine. Our study provides measurements of the growth rate of this yeast in urine with various glucose concentrations. Thus, important insights are gathered for risk group such as patients with diabetes or patients with prolonged parenteral nutrition resulting in higher urinary glucose concentration.

Microcalorimetric Method to Assess Phagocytosis: Macrophage-Nanoparticle Interactions

This study evaluated the use of isothermal microcalorimetry (ITMC) to detect macrophage-nanoparticle interactions. Four different nanoparticle (NP) formulations were prepared: uncoated poly(isobutyl cyanoacrylate) (PIBCA), polysorbate-80-coated PIBCA, gelatin, and mannosylated gelatin NPs. Changes in NP formulations were aimed to either enhance or decrease macrophage-NP interactions via phagocytosis. Alveolar macrophages were cultured on glass slabs and inserted in the ITMC instrument. Thermal activities of the macrophages alone and after titration of 100 μL of NP suspensions were compared. The relative interactive coefficients of macrophage-NP interactions were calculated using the heat exchange observed after NP titration. Control experiments were performed using cytochalasin B (Cyto B), a known phagocytosis inhibitor. The results of NP titration showed that the total thermal activity produced by macrophages changed according to the NP formulation. Mannosylated gelatin NPs were associated with the highest heat exchange, 75.4 ± 7.5 J, and thus the highest relative interactive coefficient, 9,269 ± 630 M-1. Polysorbate-80-coated NPs were associated with the lowest heat exchange, 15.2 ± 3.4 J, and the lowest interactive coefficient, 890 ± 120 M-1. Cyto B inhibited macrophage response to NPs, indicating a connection between the thermal activity recorded and NP phagocytosis. These results are in agreement with flow cytometry results. ITMC is a valuable tool to monitor the biological responses to nano-sized dosage forms such as NPs. Since the thermal activity of macrophage-NP interactions differed according to the type of NPs used, ITMC may provide a method to better understand phagocytosis and further the development of colloidal dosage forms.

Use of isothermal microcalorimetry to monitor microbial activities

Isothermal calorimetry measures the heat flow of biological processes, which is proportional to the rate at which a given chemical or physical process takes place. Modern isothermal microcalorimeters make measurements of less than a microwatt of heat flow possible. As a result, as few as 10 000-100 000 active bacterial cells in culture are sufficient to produce a real-time signal dynamically related to the number of cells present and their activity. Specimens containing bacteria need little preparation, and isothermal microcalorimetry (IMC) is a nondestructive method. After IMC measurements, the undisturbed samples can be evaluated by any other means desired. In this review, we present a basic description of microcalorimetry and examples of microbiological applications of IMC for medical and environmental microbiology. In both fields, IMC has been used to quantify microbial activity over periods of hours or even days. Finally, the recent development of highly parallel instruments (up to 48 channels) and the constantly decreasing costs of equipment have made IMC increasingly attractive for microbiology. Miniaturization of isothermal calorimeters provides an even wider range of possibilities.

Characterization of microbial activity in soil by use of isothermal microcalorimetry

Isothermal microcalorimetry is now established as a useful technique for the characterization of the microbial activity in soil. A brief summary of publications from this field and of instruments used in such work is presented. Several experimental parameters that can form important sources for systematic errors are discussed and it is suggested that further method work is made in this area. In most isothermal microcalorimetric investigations on the microbial activity in soil, the samples are amended with glucose. It is proposed that cellulose also will be used.

Use of isothermal microcalorimetry for prediction of oxidative stability of several amino acids

Isothermal microcalorimetry has been applied as a method for predicting (in)stability of ascorbic acid and several amino acids that undergo oxidative degradation in aqueous media. The fast and simple method involved the addition of different amounts of hydrogen peroxide. The appearance of the heat flow curves gave a clear general indication of how stability was influenced. The accuracy of the microcalorimetric result was investigated by comparing it with an HPLC assay and a good agreement between the results of both methods was demonstrated. It was also established that susceptibility to oxidative degradation decreases in the following order: cysteine, methionine, ascorbic acid, tyrosine and tryptophan.

Rapid Differentiation of Methicillin-Susceptible Staphylococcus aureus from Methicillin-Resistant S. aureus and MIC Determinations by Isothermal Microcalorimetry

In this study, the use of isothermal microcalorimetry (IMC) for differentiation between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) and MIC determination was evaluated. It was possible to differentiate between MRSA and MSSA within 4 h, whereas the standard method required 24 h. The MICs of cefoxitin were successfully determined for MRSA and MSSA by using IMC.

Isothermal micro-calorimetry: a tool to predict seed longevity?

This paper describes the exploratory use of isothermal micro-calorimetry (IMC) to measure directly the heat flow produced as seeds age. Heat flow was recorded in primed and non-primed (control) seeds of Ranunculus sceleratus L., aged in a micro-calorimeter at 35°C at three different seed water contents [c. 0.12, 0.075 and 0.045 g H2O (g dw)−1]. The rate of heat flow and total heat generated (an indicator of extent of reaction) were generally greater in control seeds, which aged at a faster rate, than in primed seeds. Total heat generated over a given period also increased with increasing water content. The power–time curves did not indicate first- or second-order rate kinetics, consistent with the probability that seed ageing is complex and involves a number of reactions. Even after the capacity to germinate had ceased, there was a residual power signal. As a method, IMC gave consistent results using independent samples at different times. Therefore, short-term experiments at relatively high water contents and/or temperatures may have the potential to predict the relative longevity of seed-lots, at least within a species.

Use of isothermal microcalorimetry in pharmaceutical preformulation studies, Part II. Amorphous phase quantification in a predominantly crystalline phase

Low amounts of amorphous phase present in predominantly crystalline powders were quantified by using various analytical techniques with an emphasis on the use of Isothermal Perfusion Microcalorimetry. The amorphous phase was plasticized using ethanol vapor and enthalpy of re-crystallization of amorphous phase was used for generation of a calibration curve. Amorphous content as low as 5% was quantified using this technique. Although baseline noise was very low, additional processes occurring during re-crystallization confounded quantification of lower amorphous fractions.

Assessment of Degree of Disorder (Amorphicity) of Lyophilized Formulations of Growth Hormone Using Isothermal Microcalorimetry

When determining the degree of disorder of a lyophilized cake of a protein, it is important to use an appropriate analytical technique. Differential scanning calorimetry (DSC) and X‐ray powder diffraction (XRPD) are the most commonly used thermo‐analytical techniques for characterizing freeze‐dried protein formulations. Unfortunately, these methods are unable to detect solid‐state disorder at levels < 10%. Also, interpretation of DSC results for freeze‐dried protein formulations can be difficult, as a result of the more complex thermal events occurring with this technique. For example, proteins can inhibit the thermally induced recrystallization of the lyophilized cake, resulting in potential misinterpretation of DSC degree of disorder results. The aim of this investigation was to study the use of isothermal microcalorimetry (IMC) in the assessment of degree of solid‐state disorder (amorphicity) of lyophilized formulations of proteins. For this purpose, two formulations of growth hormone were prepared by lyophilization. These formulations consisted of the same amounts of protein, mannitol, glycine, and phosphate buffer, but differed in the freeze‐drying procedure. After lyophilization, the recrystallization of the samples was studied using IMC at 25°C under different relative humidities (58–75%). The effect of available surface area was studied by determining the heat of recrystallization (Q) of the samples before and after disintegration of the cakes. The results showed that, in contrast to DSC, IMC allowed detection of the recrystallization event in the formulations. Although both formulations were completely disordered and indistinguishable according to XRPD method, IMC revealed that formulation B had a different solid‐sate structure than formulation A. This difference was the result of differences in the freeze‐drying parameters, demonstrating the importance of choosing appropriate analytical methodology.

Antimicrobial properties of silver-containing wound dressings: a microcalorimetric study

The studies reported here have been undertaken to assess the potential use of isothermal microcalorimetry in studying the antimicrobial efficacy of wound dressings that contain antimicrobial agents. The microcalorimetric technique allows non-invasive and non-destructive analysis to be performed directly on a test sample, regardless of whether it is homogeneous or heterogeneous in nature. Microcalorimetry is an established procedure that offers quantitative measurements and has the distinct advantage over traditional antimicrobial test methodologies in that calorimetric measurements are made continuously over real-time, thus the dynamic response of microorganisms to an antimicrobial agent is observed in situ. The results described in this paper are for interaction of two silver-containing wound care products AQUACEL ® Ag Hydrofiber ® (ConvaTec, Deeside, UK) and Acticoat™ 7 with SILCRYST™ (Smith and Nephew Healthcare, UK) with the wound pathogenic organisms Staphylococcus aureus and Pseudomonas aeruginosa. Both dressings are shown, microcalorimetrically, to have the capacity to kill these common wound pathogens within 1–2 h of contact. A dose–response study was conducted with the AQUACEL Ag dressing. Microcalorimetry is shown to be rapid, simple and effective in the study of the antimicrobial properties of gel forming wound dressings.

Isothermal microcalorimetry in applied biology

Techniques of isothermal microcalorimetry have been much improved during the past two decades. In addition to their use in fundamental research, applications of practical importance have been established in some areas. However, no significant use of isothermal microcalorimetry has yet been seen in practical applications of biology, despite many methodological studies reported from that area. The main problem appears to be that the sample throughput of isothermal microcalorimeters is low compared to other techniques used in that field. Further, the non-specificity of calorimetric signals is in some cases a serious limitation. Significant progress has recently been made in the design of multi-channel isothermal microcalorimeters and in techniques where specific analytical methods have been combined with isothermal microcalorimeters. Some conclusions will be drawn with respect to the use of these techniques in applied work on living materials.

Isothermal microcalorimetry and inverse phase gas chromatography to study small changes in powder surface properties

It is known that processing can alter the surface energetics of powders. In this study a sample of drug has been processed by use of different drying techniques. The samples were then assessed using inverse phase gas chromatography. It was seen that the original material had a highest surface energy and the tray-dried sample had the lowest energy surface, other samples were intermediate. The use of isothermal microcalorimetry to study water sorption to the powders revealed that the surface of the original material was unstable, as the water sorption response changed on repeat cycling. The tray-dried sample did have a stable surface which gave the same sorption response on repeat exposure to water vapour. It was concluded that the drug had minor variations in surface energy, with the as received material being in a high energy unstable state, which could be due to it being partially amorphous. The tray-dried sample had a lower energy stable surface. In certain applications differences in surface energetics could be expected to lead to changes in processing nature of the powder, so these vapour sorption techniques offer a good way of providing an assurance of the same surface energy between batches of nay material which may be at risk.

Towards More Specific Information From Isothermal Microcalorimetric Measurements on Living Systems

Calorimetry is an important thermodynamic and analytical method for investigations of living systems. The non-specific nature of calorimetry can be of advantage in such work, but will also make it difficult to interprete the results in sufficient detail. It is therefore of interest to combine calorimetry with specific analytical techniques. Work in this area is discussed with reference to the use of isothermal microcalorimetry for the characterization of living cellular systems in the pharmaceutical industry.

Using isothermal microcalorimetry for the prediction and testing of long-term properties of materials and products

The use of isothermal microcalorimetry (MC) as a sensitive monitor for slow reactions is demonstrated in a number of examples. In Example 1 the spontaneous decomposition of a nitrate ester propellant is studied and the absolute degradation rate estimated. Example 2 illustrates how MC can be used for comparing the rate of oxidative ageing at different O2 concentrations. Synergetic effects between oxygen and moisture in accelerated ageing of nitrile rubber are evaluated from a so-called 4-point test in Example 3. The last example shows a simple MC technique for rapid determination of moisture permeability through a polymeric sealant.