IP-10 Kinetics in the First Week of Therapy are Strongly Associated with Bacteriological Confirmation of Tuberculosis Diagnosis in HIV-Infected Patients.

Simple effective tools to monitor the long treatment of tuberculosis (TB) are lacking. Easily measured host derived biomarkers have been identified but need to be validated in larger studies and different population groups. Here we investigate the early response in IP-10 levels (between day 0 and day 7 of TB therapy) to identify bacteriological status at diagnosis among 127 HIV-infected patients starting TB treatment. All participants were then classified as responding or not responding to treatment blindly using a previously described IP-10 kinetic algorithm. There were 77 bacteriologically confirmed cases and 41 Xpert MTB/RIF® and culture negative cases. Most participants had a measurable decline in IP-10 during the first 7 days of therapy. Bacteriologically confirmed cases were more likely to have high IP-10 levels at D0 and had a steeper decline than clinically diagnosed cases (mean decline difference 2231 pg/dl, 95% CI: 897-3566, p = 0.0013). Bacteriologically confirmed cases were more likely to have a measurable decline in IP-10 at day 7 than clinically diagnosed cases (48/77 (62.3%) vs 13/41 (31.7%), p < 0.001). This study confirms the association between a decrease in IP-10 levels during the first week of treatment and a bacteriological confirmation at diagnosis in a large cohort of HIV positive patients.

Pyrazinamide Is Active against Mycobacterium tuberculosis Cultures at Neutral pH and Low Temperature.

For the past decades, an acidic pH has been used to render Mycobacterium tuberculosis susceptible to pyrazinamide for in vitro testing. Here, we show that at the standard breakpoint concentration and reduced culture temperatures, pyrazinamide (PZA) is active against tuberculosis (TB) at neutral pH. This finding should help unravel the mechanism of action of PZA and allow drug susceptibility testing (DST) methods to be optimized.

Current status and opportunities for therapeutic drug monitoring in the treatment of tuberculosis.

INTRODUCTION: Tuberculosis remains a global health problem and pharmacokinetic variability has been postulated as one of the causes of treatment failure and acquired drug resistance. New developments enable implementation of therapeutic drug monitoring, a strategy to evaluate drug exposure in order to tailor the dose to the individual patient, in tuberculosis treatment. AREAS COVERED: Literature on pharmacokinetics and pharmacodynamics of anti-tuberculosis drugs was explored to evaluate the effect of drug exposure in relation to drug susceptibility, toxicity and efficacy. New, down-sized strategies, like dried blood spot analysis and limited sampling strategies are reviewed. In addition, molecular resistance testing of Mycobacteria tuberculosis, combining a short turn-around time with relevant information on drug susceptibility of the causative pathogen was explored. Newly emerging host biomarkers provide information on the response to treatment. EXPERT OPINION: Therapeutic drug monitoring can minimize toxicity and increase efficacy of tuberculosis treatment and prevent the development of resistance. Dried blood spot analysis and limited sampling strategies, can be combined to provide us with a more patient friendly approach. Furthermore, rapid information on drug susceptibility by molecular testing, and information from host biomarkers on the bacteriological response, can be used to further optimize tuberculosis treatment.

New insights into the mechanism of action of pyrazinamide, implications for susceptibility testing, and future regimens.

Pyrazinamide (PZA) is included in the 2016 World Health Organization multidrug-resistant tuberculosis treatment guidelines and is a key component of most ongoing clinical trials investigating novel antibiotic combinations. PZA resistance is associated with worse tuberculosis treatment outcomes. Unfortunately, for such an important drug, phenotypic susceptibility testing is extremely challenging. The exacting bacterial growth conditions required to induce susceptibility to the drug reduce the accuracy of the susceptibility assay, even in experienced laboratories, and widespread testing is not performed. This situation is unacceptable for such a valuable and important drug. A more complete understanding of the mechanism of action of PZA would be expected to lead to improvements in this situation. Although the exact mechanism of action of PZA is not known yet, it is widely accepted that PZA is a prodrug requiring transformation to pyrazinoic acid, the active form, by the mycobacterial enzyme encoded by the pncA gene. Most clinical resistance indeed appears to be a result of a diverse range of mutations in this gene and sequencing of the pncA gene has been shown to have excellent predictive power for PZA resistance. The wider availably of pncA sequencing in combination with databases of the phenotypic implications of these mutations has helped make genetic testing for PZA resistance a practical proposition. For the past decades, it has been generally accepted that an extracellular low pH is required for PZA activity but work in our laboratory [1] and others [2] has recently challenged this assumption. Alternative bacterial stresses, apart from a reduced pH of the growth media (such as reduced temperature), can also induce a PZA-susceptible phenotype. The characterization of spontaneous in vitro-resistant pyrazinoic acid mutants selected under neutral pH conditions suggests a key role for the pantothenate/coenzyme A biosynthetic pathway. This has profound implications for the mechanism of action of PZA as well as potentially the bacterial population against which PZA is active in the host. These findings will be discussed as well as their implications for further research and the future of PZA susceptibility testing.

Cytokine Kinetics in the First Week of Tuberculosis Therapy as a Tool to Confirm a Clinical Diagnosis and Guide Therapy.

BACKGROUND: Many patients treated for tuberculosis (TB) in low and middle income countries are treated based on clinical suspicion without bacteriological confirmation. This is often due to lack of rapid simple accurate diagnostics and low healthcare provider confidence in the predictive value of current tests. We previously reported in an animal TB model that levels of host markers rapidly change in response to treatment initiation. METHODS: We assessed the potential of host biomarker kinetics of TB patients during the first two weeks of therapy to identify patients responding to treatment. Adult patients clinically diagnosed with and treated for TB, 29 in Nigeria and 24 in Nepal, were analyzed. RESULTS: Changes in concentrations of non-specific host biomarkers, particularly IP-10, in response to the first week of anti-TB therapy were strongly associated with bacteriological confirmation of TB. A decrease in IP-10 level of >300pg/ml between 0 and 7 days of treatment identified 75% of both smear-positive and smear-negative culture positive patients and correctly excluded TB in all nine culture negative patients. CONCLUSIONS: Monitoring of early IP-10 responses to treatment could form the basis of a simplified assay and could help identify patients who were erroneously clinically diagnosed with TB or those infected with drug resistant strains on inappropriate treatment. We believe this approach may be particularly appropriate for difficult to diagnose patients, e.g. smear-negative HIV-positive, or those with extra-pulmonary TB, often treated without bacterial confirmation.

Pyrazinamide resistance in Mycobacterium tuberculosis fails to bite?

In contrast to most other antimycobacterial drugs where--particularly in multidrug-resistant (MDR) strains--a limited number of resistance mutations dominate, pyrazinamide (PZA) resistance associated mutations remain highly diverse with limited clustering. This apparent lack of evolutionary selection for successful PZA resistance mechanisms deserves attention. A clear understanding of the epidemiology of PZA resistance acquisition and spread would be expected to result in important insights into how PZA might be better exploited in treatment regimens to minimize the amplification of Mycobacterium tuberculosis (MTB) drug resistance. We propose that PZA resistance typically induces a fitness cost that impairs MTB transmission. This would explain the lack of extensive clustering for PZA-resistant mutants. Our hypothesis also leads to a series of testable predictions which we outline that could confirm or refute our ideas.

Evaluation of a microcolony growth monitoring method for the rapid determination of ethambutol resistance in Mycobacterium tuberculosis.

BACKGROUND: Due to the increasing prevalence of Mycobacterium tuberculosis strains resistant to one or more antibiotics, there is a need for new quantitative culture methods both for drug susceptibility testing and for validation of mutations putatively associated with drug resistance. We previously developed a (myco) bacterial culture method, in which multiple growing microcolonies are monitored individually. Transfer of the growing microcolonies to selective medium allows the effect on the growth rate of each individual colony to be determined. As entire growing colonies are exposed to antibiotics rather than re-subbed, a second lag phase is avoided and results are obtained more rapidly. Here we investigate the performance of the microcolony method to differentiate between ethambutol (EMB) resistant, intermediate and susceptible strains. METHODS: One week old microcolonies from a reference panel of four strains with known EMB susceptibility were transferred to different concentrations of EMB. Growth rates during the 1st 2 days of exposure were used to set up classification criteria to test and classify a blinded panel of 20 tuberculosis strains with different susceptibilities. RESULTS: For 18 strains (90%) reference culture results corresponded to our classifications based on data collected within 9 days of inoculation. A single strain was classified as Intermediate instead of Susceptible, and 1 strain could not be classified due to a contamination. CONCLUSIONS: Using a microcolony growth monitoring method we were able to classify, within 9 days after inoculation, a panel of strains as EMB susceptible, intermediate or resistant with 90% correlation to the reference methods.

Early specific host response associated with starting effective tuberculosis treatment in an infection controlled placebo controlled mouse study.

Recently we proposed exploring the potential of treatment stimulated testing as diagnostic method for tuberculosis (TB). An infection controlled placebo controlled mouse study was performed to investigate whether serum cytokine levels changed measurably during the early phase of TB chemotherapy. Serum was collected prior to and during the first 3 weeks of isoniazid (INH) and rifampicin (RIF) chemotherapy, and levels of 23 selected cytokines/chemokines were measured using a liquid bead array. The serum levels of IFNγ, IP-10, MIG, MCP-1, IL-17 and IL-6 were elevated in the TB infected mice compared to non-infected mice at least at 1 time point measured. In infected mice, IFNγ, IP-10, MIG and MCP-1 levels decreased within 7 days of treatment with RIF+INH compared to placebo. Treatment of non-infected mice in the absence of tuberculosis infection had no effect on these cytokines. IL-17 and IL-6 had decreased to baseline in all infected mice prior to the initiation of treatment. This study demonstrates that systemic levels of some cytokines, more specifically IFNγ, IP-10, MIG and MCP-1, rapidly and specifically change upon starting TB chemotherapy only in the presence of infection in a mouse model. Thus, IFNγ, IP-10, MIG and MCP-1 are promising 'Treat-to-Test' targets for the diagnosis of TB and deserve further investigation in a study on human TB suspects.

Simplified automated image analysis for detection and phenotyping of Mycobacterium tuberculosis on porous supports by monitoring growing microcolonies.

BACKGROUND: Even with the advent of nucleic acid (NA) amplification technologies the culture of mycobacteria for diagnostic and other applications remains of critical importance. Notably microscopic observed drug susceptibility testing (MODS), as opposed to traditional culture on solid media or automated liquid culture, has shown potential to both speed up and increase the provision of mycobacterial culture in high burden settings. METHODS: Here we explore the growth of Mycobacterial tuberculosis microcolonies, imaged by automated digital microscopy, cultured on a porous aluminium oxide (PAO) supports. Repeated imaging during colony growth greatly simplifies "computer vision" and presumptive identification of microcolonies was achieved here using existing publically available algorithms. Our system thus allows the growth of individual microcolonies to be monitored and critically, also to change the media during the growth phase without disrupting the microcolonies. Transfer of identified microcolonies onto selective media allowed us, within 1-2 bacterial generations, to rapidly detect the drug susceptibility of individual microcolonies, eliminating the need for time consuming subculturing or the inoculation of multiple parallel cultures. SIGNIFICANCE: Monitoring the phenotype of individual microcolonies as they grow has immense potential for research, screening, and ultimately M. tuberculosis diagnostic applications. The method described is particularly appealing with respect to speed and automation.

Structure-function relationship of the human antimicrobial peptide LL-37 and LL-37 fragments in the modulation of TLR responses.

Cathelicidins are effector molecules of the innate host defense system that establish an antimicrobial barrier at epithelial interfaces. The human cathelicidin LL-37, in addition to its antimicrobial activity, also exhibits immunomodulatory effects, such as inhibition of pro-inflammatory responses to bacterial LPS in human monocytic cells. In this report, we demonstrate that LL-37 almost completely prevents the pro-inflammatory cytokine release by human peripheral blood mononuclear cells (PBMCs) following stimulation with Toll-like receptor (TLR)4 and TLR2/1 agonists while leaving TLR2/6, TLR5, TLR7 and TLR8 responses unchanged. Modulation of the TLR response by LL-37 occurred at least partly through the MAP kinase pathway via inhibition of p38 phosphorylation. By using an LL-37 library with overlapping sequences, we identified the mid-region of LL-37, comprising amino acids 13-31, as the active domain for the modulation of TLR responses. The mechanism of immunomodulation of LL-37 and LL-37 fragments is lipopoly-saccharide binding. Correlations between the capacity of LL-37 fragments to modulate TLR responses and their physico-chemical properties revealed that cationicity and hydrophobicity are essential for the modulation of LL-37-mediated TLR responses.

The human cathelicidin peptide LL-37 and truncated variants induce segregation of lipids and proteins in the plasma membrane of Candida albicans.

The human cathelicidin peptide LL-37 and several truncated variants differ in their capability to transmigrate over the plasma membrane of Candida albicans. We investigated whether retention at the cell perimeter or membrane transmigration affects their membrane-disrupting activities and candidacidal properties. Using fluorescein-labeled peptides, we demonstrate that LL-37 and its C-terminally truncated peptide LL-31 remain permanently associated with the perimeter of the cell. The N-terminally truncated peptide RK-31 initially accumulated at the cell boundary, but transmigrated into the cytoplasm within 30 min. The C-terminally truncated peptide LL-25 transmigrated instantaneously into the cytoplasm. The ultrastructural effects on the plasma membrane were studied by freeze-fracture electron microscopy combined with filipin cytochemistry. All peptides, whether they transmigrated over the plasma membrane or not, induced phase separation in the plasma membrane. All peptides induced leakage of cell components, including nucleotides and proteins. Proteins were identified by SDS-PAGE in combination with mass spectrometry, which revealed that predominantly proteins smaller than 50 kDa had leaked out of C. albicans.

Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane.

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process.

Reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5.

The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an indicator for ROS. With both peptides, a substantial enhancement of fluorescence was observed. However, TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a cell-permeant ROS scavenger, did not have an inhibitory effect on killing or on the enhancement of fluorescence. Furthermore, antimycin and azide, which have been reported to induce ROS in vitro, were not able to enhance the dihydroethidium fluorescence, while chlorhexidine, a non-specific antiseptic agent, enhanced dihydroethidium fluorescence to the same extent as did the peptides. Fluorescence microscopy showed the fluorescence enhancement to be a consequence of the release of unbound preformed ethidium from the mitochondrial matrix within the cell. It is concluded that ROS do not play a role in the histatin 5-mediated killing of C. albicans.

Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization.

A number of cationic antimicrobial peptides, among which are histatin 5 and the derived peptides dhvar4 and dhvar5, enter their target cells and interact with internal organelles. There still are questions about the mechanisms by which antimicrobial peptides translocate across the membrane. We used a liposome model to study membrane binding, translocation and membrane-perturbing capacities of histatin 5, dhvar4 and dhvar5. Despite the differences in amphipathic characters of these peptides, they bound equally well to liposomes, whereas their membrane activities differed remarkably: dhvar4 translocated at the fastest rate, followed by dhvar5, whereas the histatin 5 translocation rate was much lower. The same pattern was seen for the extent of calcein release: highest with dhvar4, less with dhvar5 and almost none with histatin 5. The translocation and disruptive actions of dhvar5 did not seem to be coupled, because translocation occurred on a much longer timescale than calcein release, which ended within a few minutes. We conclude that peptide translocation can occur through peptide-phospholipid interactions, and that this is a possible mechanism by which antimicrobial peptides enter cells. However, the translocation rate was much lower in this model membrane system than that seen in yeast cells. Thus it is likely that, at least for some peptides, additional features promoting the translocation across biological membranes are involved as well.