Mycobacterium marinum lymphocutaneous infection.

Mycobacterium marinum is a nontuberculous mycobacteria with worldwide distribution that lives in fresh or salt water and is responsible for infections in fish, and sometimes in humans. Human disease consists mainly of cutaneous nodules, but deep structure involvement may also occur. Diagnosis of M. marinum infection remains a challenge, with a considerable time delay between onset of symptoms and diagnosis. We present a 33-year-old man with no immunosuppressive history who was seen in our department with skin nodules over his hand and forearm, distributed in a sporotrichoid pattern. His hobbies included maintaining an aquarium of tropical fish. Histological examination of the patient's skin biopsy was compatible with the diagnosis of mycobacterial infection, and the Ziehl-Neelsen staining revealed acid-fast bacilli. Molecular techniques confirmed the suspicion of M. marinum infection. A necropsy was performed on one of the patient's fish, more specifically, a Poecilia reticulata, and resulted in identification of M. marinum from its gut. The patient was treated with clarithromycin, ethambutol, and rifampicin for 9 months, with clearance of infection.

Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study.

OBJECTIVES: Treatment outcome of MDR-TB is critically dependent on the proper use of second-line drugs as per the result of in vitro drug susceptibility testing (DST). We aimed to establish a standardized DST procedure based on quantitative determination of drug resistance and compared the results with those of genotypes associated with drug resistance. METHODS: The protocol, based on MGIT 960 and the TB eXiST software, was evaluated in nine European reference laboratories. Resistance detection at a screening drug concentration was followed by determination of resistance levels and estimation of the resistance proportion. Mutations in 14 gene regions were investigated using established techniques. RESULTS: A total of 139 Mycobacterium tuberculosis isolates from patients with MDR-TB and resistance beyond MDR-TB were tested for 13 antituberculous drugs: isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide, streptomycin, para-aminosalicylic acid, ethionamide, amikacin, capreomycin, ofloxacin, moxifloxacin and linezolid. Concordance between phenotypic and genotypic resistance was >80%, except for ethambutol. Time to results was short (median 10 days). High-level resistance, which precludes the therapeutic use of an antituberculous drug, was observed in 49% of the isolates. The finding of a low or intermediate resistance level in 16% and 35% of the isolates, respectively, may help in designing an efficient personalized regimen for the treatment of MDR-TB patients. CONCLUSIONS: The automated DST procedure permits accurate and rapid quantitative resistance profiling of first- and second-line antituberculous drugs. Prospective validation is warranted to determine the impact on patient care.

Screening for fragile X syndrome in males from specialized institutions in the northeast region of Brazil.

The objective of this study was to perform a study of fragile X syndrome (FXS) in São Luís, Maranhão, in males residing in five specialized institutions. Two hundred thirty-eight males with intel-lectual disability of unknown etiology participated in this study. Blood samples were processed and stored until DNA extraction. Screening for FMR1 gene mutations was performed using non-isotopic polymerase chain reaction amplification and DNA sequencing using an ABI Prism 3130 automated sequencer. Two individuals (0.84%) were positive for FMR1 mutations. One had a mutation due to expansion of the CGG repeat beyond normal levels and the other had a deletion in exon 1 of the FMR1 gene, which was confirmed by sequencing. Both probands were over 18 years old, which demonstrates the late diagnosis of the condition in these individuals and reinforces the need to implement ef-fective programs for early diagnosis of FXS in the state of Maranhão. We found that FXS might be transmitted in the families of the two indi-viduals bearing the mutation, and that it is important to understand the mutation dynamics to provide better counseling to the family members of these two individuals.

Targeting human macrophages for enhanced killing of intracellular XDR-TB and MDR-TB.

Although many compounds have been described to inhibit the replication of drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis, most of these studies only evaluate their in vitro activity. There is a lack of studies that show whether any of these agents can kill these organisms at the site where they normally reside post infection, namely, the macrophage of the lung parenchyma. It is the aim of this mini-review to identify agents that have been shown to enhance the killing of intracellular drug-susceptible, multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains by non-killing macrophages. Because these agents appear to promote their activity by affecting the transport of K(+) and Ca(2+) from the phagolysosome containing the bacteria, and thereby promoting its acidification and activation of hydrolases that will eventually kill the organism, the authors suggest that compounds that are known to affect the transport of K(+) and Ca(2+) should be considered for possible activity against intracellular MDR- and XDR-TB.

Participatory implementation of an antibiotic stewardship programme supported by an innovative surveillance and clinical decision-support system.

BACKGROUND: Antibiotic resistance will cause about 10 million deaths per year by 2050. Fighting antimicrobial resistance is a health priority. Interventions aimed to reduce antimicrobial resistance, such as antibiotic stewardship programmes (ASPs), must be implemented. To be effective, those interventions, and the implementation process, should be matched with social-cultural context. The complexity of ASPs can no longer be developed without considering both organizational and information systems. AIM: To support ASPs through the co-design and implementation, in collaboration with healthcare workers, of a surveillance and clinical decision-support system to monitor antibiotic resistance and improve antibiotic prescription. METHODS: The surveillance and clinical decision-support system was designed and implemented in three Portuguese hospitals, using a participatory approach between researchers and healthcare workers following the Design Science Research Methodology. FINDINGS: Based on healthcare workers' requirements, we developed HAITooL, a real-time surveillance and clinical decision-support system that integrates visualizations of patient, microbiology, and pharmacy data, facilitating clinical decision. HAITooL monitors antibiotic usage and rates of antibiotic-resistant bacteria, allowing early identification of outbreaks. It is a clinical decision-support tool that integrates evidence-based algorithms to support proper antibiotic prescription. HAITooL was considered valuable to support monitoring of antibiotic resistant infections and an important tool for ASP sustainability. CONCLUSION: ASP implementation can be leveraged through a surveillance and clinical decision-support system such as HAITooL that allows antibiotic resistance monitoring and supports antibiotic prescription, once it has been adapted to the context and specific needs of healthcare workers and hospitals.

Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents.

Time of Dropout From the Liver Transplant List in Patients With Hepatocellular Carcinoma: Clinical Behavior According to Tumor Characteristics and Severity of Liver Disease.

BACKGROUND: Prolonged time on the waiting list affects post-transplant survival of patients with hepatocellular carcinoma (HCC). However, it is not yet known which patients will be at higher risk for early dropout from the list. We investigate specific risk factors for early waiting list dropout in patients with HCC. METHODS: This was a single-center, intention-to-treat analysis of adults with HCC, within the Milan criteria, from July 2006 through September 2013. Patients were divided into groups according to waiting list time. The main end point was dropout from the list. RESULTS: The dropout rates of the study cohort at 3, 6, and 12-months were 6.4%, 12.4%, and 17.7%, respectively. Patients who dropped out from the list tended to be older, with blood types A and O, and with higher Child-Pugh and Model for End-Stage Liver Disease (MELD) scores. They also had larger nodules, responded poorly to trans-arterial chemo-embolization (TACE), and had a higher alpha-fetoprotein. Those with blood types B and AB appeared to be protected for dropout (odds ratio [OR] = 0.21, P = .02). Patients who responded to TACE were also protected (OR = 0.22, P < .001). When we looked into time to dropout, the only baseline characteristic that stood out was a higher MELD score (13 for those dropping out up to 90 days vs 10 for those dropping out after 180 days, P = .0025). CONCLUSIONS: We conclude that patients who drop out early from the list are primarily driven by the severity of liver disease. Patients who had progressive HCC had a high tumor load and poor response to loco-regional therapies, dropping out from the list after 180 days of inclusion.

Identification of two transcripts of canine, feline, and porcine interleukin-1 alpha.

Reverse transcription-polymerase chain reaction (RT-PCR) with interleukin-1alpha (IL-1alpha)-specific primers using total RNA from lipopolysaccharide (LPS)-stimulated lung macrophages resulted in the amplification of two distinct cDNA fragments. Cloning and sequencing of the canine and feline fragments revealed that, except for the absence of a specific 174 nucleotide sequence, the short and the long transcripts were identical. The in-frame 174 nucleotide deletion corresponds to exon 5 of the human and murine IL-1alpha gene, which encodes the cleavage site for calpain, a protein necessary for the processing of the IL-1alpha precursor into mature IL-1alpha. The two transcripts were found in the dog, cat and pig; analysis by RT-PCR, Southern and Northern blot hybridization showed no expression of the shorter IL-1alpha mRNA in equine or bovine macrophages. Expression of the two canine IL-1alpha transcripts was also detected in synovial membranes and was coordinately up-regulated in response to Borrelia burgdorferi infection under both in-vitro and in-vivo conditions.

Characterization of equine natural killer and IL-2 stimulated lymphokine activated killer cell populations.

Natural killer (NK) cells are an important component of the innate immune system. Though intensively studied in humans and rodents. NK cells remain less well characterized in other species. Studies are often limited by the lack of specific cell markers; however, the mAb NK-5C6 has been suggested to recognize an evolutionarily conserved molecule on NK cells and reacts with cells from several species. This mAb was used in the current investigation to identify and characterize equine NK cells, and was found to label approximately 10% of peripheral blood lymphocytes (PBL). Two-color flow cytometry analysis identified the NK-5C6+ cell population as being CD3-CD4- and CD8-, but positive for MHC class I and LFA-1 expression. Depletion of CD3+ T cells increased the percent NK-5C6+ cells in PBL; this enriched population demonstrated a specific cytotoxic response against a major histocompatibility complex (MHC) deficient NK target cell line (K-562), but not MHC+ target cells (EqT8888). These results provide evidence for an equine NK cell population, which exhibits endogenous lytic activity and a phenotype similar to that of human and mouse NK cells. Stimulation of peripheral blood mononuclear cells (PBMC) with IL-2 promoted the development of LAK cells. These cells were predominantly CD3+ T cells, demonstrated intracellular perforin expression, and effectively lysed both K-562 and EqT8888 target cells. Hence, equine NK cells can be identified by the NK-5C6 mAb and distinguished from IL-2 stimulated LAK cells by their cytotoxic response to specific target cell lines.

ACTH treatment disrupts ovarian IGF-I and steroid hormone production.

Hyper-adrenal activity and increased glucocorticoid hormone release are associated with disruptions in reproductive function and adverse effects on the ovary. The aim of this investigation was to determine whether elevated glucocorticoid hormone levels can influence ovarian IGF-I synthesis and action in vivo. To elevate endogenous glucocorticoid levels, gilts were treated with ACTH during the luteal phase of the oestrous cycle (days 9-13) while the control group received saline. The gilts were subsequently ovariectomized on either day 14 or day 18 of the oestrous cycle. Follicular fluid (FF) was collected from individual follicles; IGF-I and steroid hormone concentrations were determined by radioimmunoassay, and IGF-binding protein (IGFBP) expression was assessed by Western ligand blotting. Granulosa cells were also recovered and placed in culture to determine IGF-I, progesterone (P(4)) and oestradiol-17beta (E(2)) production levels. The cells were cultured in serum-free medium for 5 days and supplemented with: (a) media alone, (b) IGF-I, (c) FSH and androstenedione (A(4)), or (d) IGF-I with FSH and A(4). The FF from ACTH-treated gilts was characterized by elevated (P<0.05) cortisol levels on day 14 and lower (P<0.05) E(2) values on both day 14 and day 18. Lower (P<0.05) IGF-I concentrations were also measured in the FF of ACTH-treated gilts collected on day 18. This altered hormone profile in FF was associated with impaired IGF-I and steroid hormone synthesis by granulosa cells. IGF-stimulated P(4) production (P<0.01) by cells recovered from ACTH-treated gilts on day 14 was lower (P<0.05). By day 18, IGF-I, P(4) and E(2) production by cells from the ACTH group were all significantly (P<0. 05) lower. These results demonstrate that increased glucocorticoid concentrations can disrupt subsequent ovarian IGF-I synthesis and IGF action in vivo and can, potentially, impair follicle maturation.

Structural and functional analysis of the gene cluster encoding carotenoid biosynthesis in Mycobacterium aurum A+.

The fragment containing the carotenoid gene cluster from Mycobacterium aurum A+, a 3,3'-dihydroxy-isoneriatene and 3-monohydroxy-isoneriatene accumulator, has been sequenced and the exposed eight genes are organised in two operons. The function of three of these genes, a phytoene desaturase (crtI), a phytoene cyclase (crtY) and a beta-carotene desaturase (crtU), was demonstrated by complementation of M. aurum carotenoid mutants. The eight genes of the carotenoid cluster are highly homologous to other carotenoid gene clusters and thus this cluster is a candidate for its introduction into mycobacteria as a non-antibiotic reporter gene(s) as well as a source of new regulated promoters.

Evaluation of a commercial ligase chain reaction assay for the diagnosis of pulmonary and extra-pulmonary tuberculosis.

SETTING: Egas Moniz Hospital, Lisbon, Portugal. OBJECTIVE: To evaluate the Ligase Chain Reaction (LCx) Mycobacterium tuberculosis Assay for the direct detection of M. tuberculosis complex in respiratory specimens after smear observation, and its suitability for non-respiratory clinical specimens. DESIGN: Analysis of 156 specimens collected from 123 patients with pulmonary tuberculosis and/or extrapulmonary involvement. RESULTS: Among 93 pulmonary secretions and 63 extra-pulmonary samples and after resolution of discrepancies based on clinical and laboratory findings, two pulmonary samples from a patient with a diagnosis of sarcoidosis, four samples of cerebrospinal and one of seminal fluid were considered as false positives. Two tissue biopsy samples, one pericardial effusion and one pulmonary secretion from patients strongly suspected of having tuberculosis were considered as false negatives for the assay, without inhibition of amplification. All specimens yielding M. avium on culture were LCx negative. CONCLUSION: The LCx Mycobacterium tuberculosis Assay was found to be useful for the rapid identification of M. tuberculosis complex in all types of specimens. It revealed a high specificity both in pulmonary and extrapulmonary products, and a sensitivity of 97% for the pulmonary secretions and of 75% for the extra-pulmonary specimens, independently of the bacilloscopy results.

Outbreak of multiple drug-resistant tuberculosis in Lisbon: detection by restriction fragment length polymorphism analysis.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) mainly among human immunodeficiency virus (HIV) seropositive patients in Lisbon hospitals in 1996-1997. OBJECTIVE: Detection of transmission of MDR-TB strains and epidemic outbreaks in several hospital units in the city of Lisbon, including a prison hospital. DESIGN: Use of restriction fragment length polymorphism (RFLP) to fingerprint isolates of Mycobacterium tuberculosis resistant to isoniazid, rifampicin, and one other drug. RESULTS: A total of 43 MDR-TB strains were typed. Sixty-seven per cent of the patients were HIV positive, 12% were HIV negative, and the remainder had unknown HIV status. About 88% of the isolates were grouped in three genetically similar clusters, suggesting possible recent transmission. A predominant cluster (cluster A), corresponding to 72% of the cases, was found, 45% of which came from the prison hospital. Strains from this cluster were resistant to isoniazid, rifampicin, streptomycin, and sometimes ethambutol. A retrospective epidemiological investigation was conducted with respect to all patients in cluster A, and epidemiological links were established between them. CONCLUSION: Our results suggest recent transmission of MDR-TB, mainly in HIV-positive patients, in Lisbon hospitals. Moreover, the predominant MDR-TB clustered strains were not confined to HIV-infected individuals, as they were also isolated in some immunocompetent patients.

Enhancement of antibiotic activity against poly-drug resistant Mycobacterium tuberculosis by phenothiazines.

Phenothiazines have been shown to inhibit the in vitro growth of multi-drug resistant (resistant to rifampicin and isoniazid) Mycobacterium tuberculosis (MDRTB). They have been considered as potential adjuvants to regimens employing four or more antibiotics for the management of freshly diagnosed infections of M. tuberculosis in patients from areas known to have a high prevalence of MDRTB. Chlorpromazine has been shown to enhance the activity of antibiotics (except ethambutol) to which M. tuberculosis is susceptible. This might result in a reduction in the dose of some or all of the antibiotics employed without sacrificing the integrity of treatment. Chlorpromazine, thioridazine and promethazine were shown to enhance the activity of rifampicin and streptomycin when used in combinations at concentrations that are minimally effective when employed separately against clinical strains of M. tuberculosis resistant to two or more antibiotics (poly-drug resistant MTB). The phenothiazines had no effect on the activity of isoniazid against poly-drug resistant MTB.

Glucocorticoid influence on porcine granulosa cell IGF-I and steroid hormone production in vitro.

The effect of cortisol on granulosa cell (GC) insulin-like growth factor I (IGF-I) synthesis, and IGF-mediated steroid production was examined at various stages of follicle maturation. Granulosa cells were recovered from gilts on Days 14, 18, and 20 of the estrous cycle, while luteinizing GC were recovered on Day 21, just prior to ovulation. The cells were cultured in serum-free medium with increasing concentrations of cortisol (0, 1, 10, and 100 microg/mL) for 5 d with or without IGF-I stimulation (10 ng/mL). During culture all cells were supplemented with FSH and androstenedione (A4). Cellular IGF-I, progesterone (P4) and estradiol-17beta (E2) production was determined by specific radioimmunoassays (RIA), and cell proliferation was assessed. Granulosa cell IGF-I and steroid hormone synthesis increased (P<0.05) with follicle maturation. Direct exposure to high cortisol concentrations, however, altered both IGF-I synthesis and action. Cortisol treatment lowered (P<0.05) IGF-I production by GC recovered on Days 18, 20, and 21. Furthermore, it reduced (P<0.05) IGF-stimulated P4 synthesis at all stages and decreased (P<0.05) IGF-stimulated E2 synthesis by cells recovered on Day 14. In contrast, cortisol enhanced (P<0.05) FSH-stimulated P4 production by GC collected on Days 14 and 18. The opposing effects on FSH and IGF-I action indicate that cortisol did not promote an overall suppressive effect on cell function, nor did it impair cell proliferation. Hence, these results demonstrate that elevated cortisol concentrations can disrupt both IGF-I synthesis and IGF-mediated actions by porcine GC under in vitro conditions, and that specific disruptions are dependent on the stage of follicle maturation.

Rate of decline of cortisol concentrations in ovarian follicles following ACTH treatment in the sow.

The rates of decline in cortisol concentrations in blood and ovarian follicular fluid were assessed in cyclic sows (n = 30) after treatment with saline or a depot form of adrenocorticotrophic hormone (ACTH). After a single injection of ACTH (0.5 iu/kg, BW, i.m.), peak cortisol values were achieved in blood within 3 to 4 h followed by a half-life net clearance rate (t1/2 of 2.40 +/- 0.29 (SE) h. The same dose of ACTH was then given at 12 h intervals from days 9 to 13 of the estrous cycle. On day 14 the concentrations of cortisol in follicular fluid were higher (P < 0.05) in ACTH-injected sows than in saline-injected controls. A t1/2 value of 37.81 h was determined for cortisol based on the decline in concentrations in follicular fluid collected on days 14, 16 and 18. This relatively slow rate of removal from developing ovarian follicles may have implications for the previously observed detrimental effects of increased cortisol concentrations on follicular development.

GidB mutation as a phylogenetic marker for Q1 cluster Mycobacterium tuberculosis isolates and intermediate-level streptomycin resistance determinant in Lisbon, Portugal.

Development of streptomycin resistance in Mycobacterium tuberculosis is usually associated with mutations in rpsL and rrs genes, although up to 50% of clinical streptomycin-resistant isolates may present no mutation in either of these genes. In the present report we investigate the role of gidB gene mutations in streptomycin resistance. We have analyzed 52 streptomycin-resistant and 30 streptomycin-susceptible Mycobacterium tuberculosis clinical isolates by sequencing and endonuclease analysis of the gidB and rpsL genes. All clinical isolates were genotyped by 12-loci MIRU-VNTR. The gidB gene of 18 streptomycin-resistant isolates was sequenced and four missense mutations were found: F12L (1/18), L16R (18/18), A80P (4/18) and S100F (18/18). The remaining isolates were screened by endonuclease analysis for mutations A80P in the gidB gene and K43R in the rpsL gene. Overall, mutation A80P in the gidB gene was found in eight streptomycin-resistant isolates and 11 streptomycin-susceptible multidrug-resistant isolates. Also noteworthy, is the fact that gidB mutations were only present in isolates without rpsL and rrs mutations, all from genetic cluster Q1. Streptomycin quantitative drug susceptibility testing showed that isolates carrying the gidB A80P mutation were streptomycin intermediate-level resistant and that standard drug susceptibility testing yielded inconsistent results, probably due to borderline resistance. We conclude that gidB mutations may explain the high number of streptomycin-resistant strains with no mutation in rpsL or rrs. These mutations might occasionally confer low-level streptomycin resistance that will go undetected in standard susceptibility testing.

Antibacterial properties of compounds isolated from Carpobrotus edulis.

Several compounds isolated from the plant Carpobrotus edulis were evaluated for their activity against multidrug-resistant (MDR) bacteria and their efflux pump systems. Amongst the compounds isolated, six compounds were tested, namely uvaol, ß-amyrin, oleanolic acid, catechin, epicatechin and monogalactosyldiacylglycerol. Oleanolic acid presented high antibacterial activity against a large number of bacterial strains. The triterpene uvaol was the most active compound for modulation of efflux activity by MDR Gram-positive strains.