Microcolonies in fluoroquinolone agar proportion susceptibility testing of Mycobacterium tuberculosis: an indicator of drug resistance.

Microcolony growth of Mycobacterium tuberculosis on agar proportion susceptibility testing is neither well-defined nor previously reported with fluoroquinolone susceptibility testing. We describe here M. tuberculosis microcolony growth with fluoroquinolones, and assess its clinical significance. We screened 797 M. tuberculosis isolates for ofloxacin resistance (2.0 µg/mL) by agar proportion; 19 ofloxacin-resistant and 38 ofloxacin-susceptible isolates were selected for more detailed susceptibility testing with ofloxacin, ciprofloxacin, levofloxacin (all at 2.0 µg/mL) and moxifloxacin (0.5 µg/mL). The 57 isolates were also tested at two concentrations both above and below the critical concentrations. Microcolonies were defined as colonies 0.2-0.4 mm in diameter; confirmed microcolonies were present on repeat testing. Of the 57 isolates tested in detail, 7 grew microcolonies, of which 2 (0.3% of all isolates tested) had confirmed microcolonies on repeat testing (6 tests performed, and microcolonies were present on at least 4). Both M. tuberculosis isolates were ofloxacin-resistant on screening, and had ofloxacin minimum inhibitory concentration (MIC) >8 µg/mL. The five other isolates were ofloxacin-susceptible on screening, but had regular colony growth (i.e., resistance) at the drug concentration that initially resulted in microcolonies (ofloxacin 0.5 or 1.0 µg/mL). Microcolonies were observed infrequently with fluoroquinolone susceptibility testing, but when confirmed, they were associated with drug resistance.

Biomarkers of systemic inflammation predict survival with first-line immune checkpoint inhibitors in non-small-cell lung cancer.

INTRODUCTION: Pembrolizumab is an established first-line option for patients with advanced non-small-cell lung cancer (NSCLC) expressing programmed death-ligand 1 ≥50%. Durable responses are seen in a subset of patients; however, many derive little clinical benefit. Biomarkers of the systemic inflammatory response predict survival in NSCLC. We evaluated their prognostic significance in patients receiving first-line pembrolizumab for advanced NSCLC. METHODS: Patients treated with first-line pembrolizumab for advanced NSCLC with programmed death-ligand 1 expression ≥50% at two regional Scottish cancer centres were identified. Pretreatment inflammatory biomarkers (white cell count, neutrophil count, neutrophil/lymphocyte ratio, platelet/lymphocyte ratio, albumin, prognostic nutritional index) were recorded. The relationship between these and progression-free survival (PFS) and overall survival (OS) were examined. RESULTS: Data were available for 219 patients. On multivariate analysis, albumin and neutrophil count were independently associated with PFS (P < 0.001, P = 0.002, respectively) and OS (both P < 0.001). A simple score combining these biomarkers was explored. The Scottish Inflammatory Prognostic Score (SIPS) assigned 1 point each for albumin <35 g/l and neutrophil count >7.5 × 109/l to give a three-tier categorical score. SIPS predicted PFS [hazard ratio 2.06, 95% confidence interval (CI) 1.68-2.52 (P < 0.001)] and OS [hazard ratio 2.33, 95% CI 1.86-2.92 (P < 0.001)]. It stratified PFS from 2.5 (SIPS2), to 8.7 (SIPS1) to 17.9 months (SIPS0) (P < 0.001) and OS from 5.1 (SIPS2), to 12.4 (SIPS1) to 28.7 months (SIPS0) (P < 0.001). The relative risk of death before 6 months was 2.96 (95% CI 1.98-4.42) in patients with SIPS2 compared with those with SIPS0-1 (P < 0.001). CONCLUSIONS: SIPS, a simple score combining albumin and neutrophil count, predicts survival in patients with NSCLC receiving first-line pembrolizumab. Unlike many proposed prognostic scores, SIPS uses only routinely collected pretreatment test results and provides a categorical score. It stratifies survival across clinically meaningful time periods that may assist clinicians and patients with treatment decisions. We advocate validation of the prognostic utility of SIPS in this and other immune checkpoint inhibitor treatment settings.

Immunocytochemical identification of interstitial cells of Cajal in the murine fundus using a live-labelling technique.

Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells.

Pilot study to examine the effect of antibiotic therapy on MRI outcomes in RRMS.

This trial examined the safety and possible MRI and clinical effects of anti-chlamydial antibiotic therapy in relapsing-remitting MS (RRMS). Newly diagnosed MS patients were selected to participate if they showed Chlamydia pneumoniae gene in their CSF and had one or more enhancing lesions on brain magnetic resonance imaging (MRI). After a 4-month run in phase of monthly MRI, patients were randomized to receive rifampin (300 mg twice daily) and azithromycin (500 mg every other day) for 6 months or placebo (PBO). Patients then had monthly MRI on therapy and two additional scans on months 12 and 14. Lumbar punctures were repeated between months 7 and 8 and within 2 weeks of termination of the study. Data on 4 patients on treatment and 4 on PBO were available for analysis. The primary outcome measure of showing a beneficial effect on enhancing lesions was not met. However, there was a significant difference in brain parenchymal fraction loss favoring those patient receiving antibiotics compared with PBO (p< or =0.02). Three of the four patients on antibiotic therapy cleared the organism from the CSF by month 12; in the PBO group one patient cleared the organism. The reduction in atrophy in patients receiving antibiotics must be viewed with caution, due to the small number of patients studied.

Regulation by IFN-beta of inducible nitric oxide synthase and interleukin-12/p40 in murine macrophages cultured in the presence of Chlamydia pneumoniae antigens.

Chlamydia pneumoniae has been demonstrated in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS). Interferon-beta (IFN-beta) has favorable effects on the clinical course of MS. We investigated whether the beneficial effects of IFN-beta in MS may involve its role in regulating nitric oxide (NO) and interleukin-12 (IL-12) in macrophages, as these immune modulators form part of the innate immune response to intracellular pathogens, such as C. pneumoniae. Murine macrophages in cultures exposed to elementary body antigens or recombinant major outer membrane protein (rMOMP) of C. pneumoniae demonstrate a significant increase in NO as well as production of IL-12/p40 in culture supernatants compared with basal levels. Addition of murine IFN-beta increased NO activity in murine macrophages cultured with chlamydial antigens. Addition of neutralizing anti-IFN-beta antibody prevented the NO increase. In contrast to its effect on inducible NO synthase (iNOS), IFN-beta reduced induction of IL-12/p40 following culture with either elementary body antigens or rMOMP. Inhibition was reversed with anti-IFN-beta antibody. If C. pneumoniae infection is responsible for the inflammatory response in the pathogenesis of MS, the beneficial effects of IFN-beta in MS may be due to its enhancing intracellular NO activity while inhibiting secretion of the proinflammatory cytokine, IL-12.

CSF oligoclonal bands in MS include antibodies against Chlamydophila antigens.

BACKGROUND: Considerable evidence suggests the role of an infectious agent in MS. The presence of Chlamydophila pneumoniae in CSF from patients with MS was shown earlier; to further examine this association the reactivity of the oligoclonal antibody response in the CSF of patients with MS to C pneumoniae antigens was determined and compared with other antigens. METHODS: Seventeen patients with MS and 14 control subjects with other neurologic disease were studied. Affinity-driven immunoblot studies and solid-phase adsorption of CSF oligoclonal bands by elementary body antigens of C pneumoniae, viral antigens (measles and herpes simplex virus-1), bacterial antigen (Escherichia coli and Staphylococcus aureus), and heat shock protein-60 were performed. RESULTS: Affinity-driven immunoblot studies demonstrated reactivity of oligoclonal bands in CSF samples from 16 patients with MS against elementary body antigens of C pneumoniae. None of the control subjects showed a prominent reactivity to elementary body antigens of C pneumoniae. In 14 of 17 patients with MS examined, oligoclonal bands were adsorbed either partially or completely from the CSF by elementary body antigens of C pneumoniae, but not by myelin basic protein, heat shock protein-60, or bacterial or viral antigens. In three patients with subacute sclerosing panencephalitis, adsorption of oligoclonal bands was seen with measles virus antigens but not with elementary body antigens of C pneumoniae. CONCLUSIONS: Oligoclonal bands in CSF of patients with MS include antibodies against Chlamydophila antigens.

Intraventricular morphine administration for control of chronic cancer pain.

Twenty cancer patients with severe chronic pain have been treated with intraventricular morphine sulfate. Adequate pain relief until death was achieved in 10 patients; 1 patient has been treated for 9 months and is still being treated. In 2 patients, the effects of the morphine sulfate on their unilateral pelvic pain wore off after 4 and 6 months because of tumor progression. At that time, they underwent chordotomy procedures elsewhere. The treatment was discontinued in 4 patients for reasons other than inadequate pain relief, such as medical complications or resolution of pain. In 3 patients, the procedure was abandoned when emotional and psychological factors interfered with pain control. Dose requirements of intraventricular morphine sulfate varied greatly, depending on the total daily dose of systemic narcotic intake at the onset of the study. Intraventricular morphine sulfate is a feasible and reliable method to achieve pain relief in selected cancer patients with severe chronic pain when the maximum tolerated dose of systemic narcotic analgesics has become insufficient to control their pain.

Tuberculoid tenosynovitis and carpal tunnel syndrome caused by Mycobacterium szulgai.

Mycobacterium szulgai, a scotochromogenic mycobacterium, is a newly recognized pathogen of man and has been reported to cause pulmonary infections, olecranon bursitis and cervical adenitis. We isolated M. szulfai from granulomatous tissue removed at surgery from a young florist with the carpal tunnel syndrome. The organism was susceptible to ethambutol and rifampin but resistant to isoniazid. Cure was achieved by debridement and chemotherapy with ethambutol and rifampin. Neither the source in our patient nor the natural habitat of M. szulgai is known. Because it resembles M. gordonae and M. flavescens, common scotochromogenic mycobacteria in tapwater, care must be taken to avoid dismissing M. szulgai as a contaminant when it is isolated from tissue.

Spontaneous bacterial peritonitis. A review of 28 cases with emphasis on improved survival and factors influencing prognosis.

During a five year period, 28 episodes of spontaneous bacterial peritonitis were documented. The number of cases recognized annually increased during the study period. Clinical and laboratory features of spontaneous bacterial peritonitis were similar to those previously reported; however, mortality was considerably lower (57 per cent). Factors associated with adverse prognosis were increasing hepatic encephalopathy, more than 85 per cent granulocytes in peripheral blood or ascitic fluid, total bilirubin greater than 8 mg/dl and serum albumin less than 2.5 g/dl. Temperature greater than 38 degrees C was associated with increased survival. Infection by enteric organisms was associated with higher mortality than infection by nonenteric organisms. Unexpectedly, patients with bacteremia fared no worse than those whose blood remained sterile. The data suggest that in patients with leukocyte counts greater than 1,000 cells/mm3 and more than 85 per cent granulocytes in their ascitic fluid, the likelihood of spontaneous bacterial peritonitis is high. Such patients deserve empiric antibiotic therapy pending the results of appropriate cultures.

Granulomatous tenosynovitis and carpal tunnel syndrome caused by Sporothrix schenckii.

Although the usual form of sporotrichosis is a lymphocutaneous lesion, Sporothrix schenckii can cause articular disease that is difficult to diagnose. We describe two patients with sporotrichosis who presented with tenosynovitis and the carpal tunnel syndrome. A tissue specimen is required for a precise diagnosis; granulomatous tenosynovitis suggests an infectious cause. Unless appropriate cultures for bacteria, mycobacteria and fungi are obtained, the diagnosis may be missed or delayed. Mouse inoculations may be required to isolate S. schenckii from tissue, which rarely shows the delicate fungus in histologic sections. Our patients were cured by a combination of synovectomy and the intravenous administration of amphotericin B. Sporotrichosis should be considered in the differential diagnosis of the carpal tunnel syndrome, particularly when surgical exploration discloses a granulomatous tenosynovitis.

Multicenter collaborative evaluation of a standardized serum bactericidal test as a predictor of therapeutic efficacy in acute and chronic osteomyelitis.

Forty-eight episodes of osteomyelitis, 30 acute and 18 chronic, were evaluated in a prospective multicenter collaborative study to determine whether a standardized serum bactericidal test could predict outcome of infection. All centers used a microdilution test method that defined the recognized important test variables, including inoculum size, culture medium, dilution technique, incubation time, method of subculture, and bactericidal endpoint. In patients with acute osteomyelitis, peak serum bactericidal titers had no predictive value; however, trough titers of 1:2 or greater accurately predicted cure, whereas trough titers of less than 1:2 predicted therapeutic failure. In patients with chronic osteomyelitis, peak serum bactericidal titers of 1:16 or greater and trough titers of 1:4 or greater accurately predicted cure, whereas peak titers of less than 1:16 and trough titers of less than 1:2 accurately predicted failure. It is concluded that this standardized serum bactericidal test provides good prognostic information in patients with osteomyelitis, and it is recommended that patients with acute osteomyelitis have serum bactericidal titers of 1:2 or greater at all times and that patients with chronic osteomyelitis have serum bactericidal titers of 1:4 or greater at all times.

Multicenter collaborative evaluation of a standardized serum bactericidal test as a prognostic indicator in infective endocarditis.

One hundred twenty-nine patients with bacterial endocarditis were evaluated in a multicenter collaborative study to determine whether a standardized serum bactericidal test could predict the outcome of the infection. All centers used a microdilution test method that defined all known test variables, including inoculum size, culture medium, dilution technique, incubation time, method of subculture, and bactericidal endpoint. Peak serum bactericidal titers of 1:64 or more and trough serum bactericidal titers of 1:32 or more predicted bacteriologic cure in all patients. The traditionally recommended serum bactericidal titer of 1:8 had statistically significant predictive accuracy at trough antibiotic levels only. The serum bactericidal test was a poor predictor of bacteriologic failure and ultimate clinical outcome, which depends on many factors. Wider recognition by physicians and clinical microbiologists that this in vitro test of antimicrobial activity can accurately predict bacteriologic success but cannot accurately predict either bacteriologic failure or clinical outcome could lead to a better consensus about its appropriate use. On the basis of the results of this study, peak serum bactericidal titers of 1:64 or more and trough serum bactericidal titers of 1:32 or more are recommended to provide optimal medical therapy for infective endocarditis.

Inhibitors of cholesterol biosynthesis. 1. trans-6-(2-pyrrol-1-ylethyl)-4-hydroxypyran-2-ones, a novel series of HMG-CoA reductase inhibitors. 1. Effects of structural modifications at the 2- and 5-positions of the pyrrole nucleus.

A novel series of trans-6-(2-pyrrol-1-ylethyl)-4-hydroxypyran-2-ones and their dihydroxy acid derivatives were prepared and evaluated for their ability to inhibit the enzyme HMG-CoA reductase in vitro. A systematic study of substitution at the 2- and 5-positions of the pyrrole ring revealed that optimum potency was realized with the 2-(4-fluorophenyl)-5-isopropyl derivative 8x, which possessed 30% of the in vitro activity of the potent fungal metabolite compactin (I). A molecular modeling analysis led to the description of a pharmacophore model characterized by (A) length limits of 5.9 and 3.3 A for the 2- and 5-substituents, respectively, as well as an overall width limit of 10.6 A across the pyrrole ring from the 2- to the 5-substituent and (B) an orientation of the ethyl(ene) bridge to the 4-hydroxypyran-2-one ring nearly perpendicular to the planes of the parent pyrrole, hexahydronaphthalene, and phenyl rings of the structures examined (Figure 3, theta = 80-110 degrees). Attempts to more closely mimic compactin's polar isobutyric ester side chain with the synthesis of 2-phenylpyrroles containing polar phenyl substituents resulted in analogues with equal or slightly reduced potencies when compared to the 2-[(unsubstituted or 4-fluoro)phenyl]pyrroles, supporting the hypothesis that inhibitory potency is relatively insensitive to side-chain polarity or charge distribution in this area.

Synthesis and antiarrhythmic activity of cis-2,6-dimethyl-alpha,alpha-diaryl-1-piperidinebutanols.

A series of alpha,alpha-diaryl-1-piperidinebutanols was evaluated for antiarrhythmic activity in the coronary ligated dog model. Structure-activity relationship studies indicated that the 2,6-dimethylpiperidine group yielded compounds with the best antiarrhythmic profiles in this series. The length of the methylene chain separating the diarylcarbinol and the amino group was not crucial. Substitution of a hydrogen or a number of functional groups for the hydroxyl group had little effect on efficacy or duration but yielded compounds that produced severe tachycardias. Replacement of one of the aryl groups by hydrogen or a pyridinyl or cyclohexyl group had little effect on efficacy but decreased the duration of action. Compound 18 (pirmenol) was ultimately chosen for further studies and is now being investigated in man.

Cardioselective beta-adrenergic blocking agents. 1. 1-((3,4-Dimethoxyphenethyl)amino)-3-aryloxy-2-propanols.

A series of 1-amino-3-aryloxy-2-propanols has been synthesized and examined for cardioselective beta-blockade. The introduction of the (3,4-dimethoxyphenethyl)amino group lead to the most cardioselective agents. Structure-activity relationships are discussed. Of the compounds tested 1-[(3,4-dimethoxyphenethyl)amino]-3-(m-tolyloxy)-2-propanol was selected for clinical trial because of optimal potency and selectivity.

Inhibitors of cholesterol biosynthesis. 3. Tetrahydro-4-hydroxy-6-[2-(1H-pyrrol-1-yl)ethyl]-2H-pyran-2-one inhibitors of HMG-CoA reductase. 2. Effects of introducing substituents at positions three and four of the pyrrole nucleus.

A series of trans-tetrahydro-4-hydroxy-6-[2-(2,3,4,5-substituted-1H-pyrrol-1-yl) ethyl]-2H-pyran-2-ones and their dihydroxy acids were prepared and tested for their ability to inhibit the enzyme HMG-CoA reductase in vitro. Inhibitory potency was found to increase substantially when substituents were introduced into positions three and four of the pyrrole ring. A systematic exploration of structure-activity relationships at these two positions led to the identification of a compound ((+)-33,(+)-(4R)-trans-2-(4-fluororphenyl)-5-(1-methylethyl)-N,3- diphenyl-1- [(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1H-pyrrole-4- carboxamide) with five times the inhibitory potency of the fungal metabolite compactin.

Synthesis and antiarrhythmic activity of alpha-[(diarylmethoxy)methyl]-1-piperidineethanols.

A series of alpha-[(diarylmethoxy)methyl]-1-piperidineethanols was evaluated for antiarrhythmic activity in the coronary artery ligated dog model. Structure-activity relationship studies indicated that the 2,6-dimethylpiperidine group afforded the best antiarrhythmic agents in this series and was essential for long duration of action. This investigation indicated that quaternary ammonium salts were not essential for a long duration of action. It was also shown that the antiarrhythmic activity could be separated from the tachycardia frequently caused by this type of agent.

The problem of selecting antimicrobial agents before (or without) the results of susceptibility testing.

The appropriate selection of an antimicrobial regimen depends upon more factors than merely the results of susceptibility testing. It is a task more difficult today because of the recent development of many new antimicrobial agents. Clinicians who prescribe antibiotics need to invest time and energy beyond reading a package insert in order to gain familiarity with antimicrobial agents. Understanding the antimicrobial and pharmacologic properties of a few agents from each class of antimicrobial agents will help make the selection of empiric regimens less difficult. Essential to empiric therapy is frequent and habitual reassessment of antimicrobial regimens with possible application of cost-effective modifications kept in mind. Once the pathogen is isolated, the empiric regimen can be modified. The very last factor that is used in modification of the antimicrobial regimen is the result of susceptibility testing. Prior to (or without) this information, the clinical outcome of serious infections most often will depend upon the early institution of appropriate empiric therapy.

Tuberculosis, infection control, and the microbiology laboratory.

The diagnosis of tuberculosis is an increasingly important problem confronting the clinical microbiology laboratory as well as hospital epidemiologists. The inherent delays in conventional laboratory procedures for the isolation and susceptibility testing of M tuberculosis may contribute directly to the spread of tuberculosis. Application of newer methods that are more rapid is essential if the spread of tuberculosis is to be limited. Hospital epidemiologists and clinical microbiology laboratories should work together to critically examine all of their current procedures for tuberculosis and take steps to correct or improve all omissions and delays in mycobacterial tests ordering, collection, testing, and reporting.