Efficient Differentiation of Mycobacterium avium Complex Species and Subspecies by Use of Five-Target Multiplex PCR

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.Since the early 1980s, there has been an increase in disease caused by organisms broadly categorized as nontuberculous mycobacteria (NTM), a generic term for mycobacteria not in the Mycobacterium tuberculosis complex and other than M. leprae (32). Of these NTM, Mycobacterium avium complex (MAC) species are the most common cause of human and animal disease globally (6, 14, 16, 24). The clinical relevance of the MAC in humans has been amplified in recent decades with the increasing population of immunocompromised individuals resulting from longer life expectancy, immunosuppressive chemotherapy, and the AIDS pandemic (27). The MAC is divided into two main species: M. avium and M. intracellulare. M. avium is further subdivided (per Turenne et al.) into four subspecies: M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum (39).Members of the family Mycobacteriaceae, comprising the MAC, differ in virulence and ecology. Those designated M. avium subsp. hominissuis are genomically diverse, low-virulence, opportunistic pathogens for both animals and humans. The majority of human M. avium subsp. hominissuis infections occur in HIV-immunocompromised people, immunocompetent persons with underling pulmonary disease, and children with cystic fibrosis (2, 12, 17). Considered ubiquitous in the environment (the most likely source of infection for humans), M. avium subsp. hominissuis has been isolated from water, soil, and dust (9). Domestic water distribution systems have been reported as possible sources of M. avium subsp. hominissuis infections in hospitals, homes, and commercial buildings (26, 27). In animals, M. avium subsp. hominissuis is found as a cause of lymphadenitis of the head and mesenteric lymph nodes of swine recognized at slaughter.Mycobacterium avium subsp. avium has long been recognized as a primary pathogen causing avian tuberculosis in wild and domestic birds (37, 38). Members of this subspecies also sporadically cause disease in other animals (6, 15, 30).For veterinarians, the MAC member of greatest importance is M. avium subsp. paratuberculosis. This MAC member causes a chronic granulomatous enteritis called Johne''s disease or paratuberculosis, most often in ruminants (16, 22, 31). Mycobacterium avium subsp. paratuberculosis is capable of infecting and causing disease a wide array of animal species, including nonhuman primates, without need of immunosuppressive coinfections. The herd-level prevalence of M. avium subsp. paratuberculosis infections in dairy cattle exceeds 50% in most major dairy product-producing countries (29, 31). Two systematic reviews and meta-analyses report a consistent association of M. avium subsp. paratuberculosis with Crohn''s disease, and the zoonotic potential of M. avium subsp. paratuberculosis continues to be a controversial subject discussed in the literature (1, 11). Unlike for most other M. avium subspecies, isolation of M. avium subsp. paratuberculosis requires the addition of the siderophore mycobactin to culture media and prolonged culture incubation for successful isolation from a tissue, soil, or fecal samples (43). After this lengthy incubation period with special media, resultant acid-fast organisms then need to be accurately identified.Unlike the M. avium subspecies, whose type strains were obtained from nonhuman hosts, the type strain of M. intracellulare (ATCC 13950) was isolated from a human, specifically a child who died from disseminated disease. Recently, numerous isolates considered to be M. intracellulare were reclassified as M. chimaera sp. nov. as part of the MAC (35). Few of these isolates were found to be clinically relevant, suggesting that this MAC species has low pathogenicity, and this factor is crucial to therapeutic decision making. Mycobacterium intracellulare appears to have a distinct environmental niche, more prevalent in biofilms and at significantly higher CFU numbers than M. avium (10, 36). It accounts for more documented human infections than M. avium subsp. hominissuis in several countries, including South Korea and Japan (19, 20, 23).Contemporary methods for MAC identification, e.g., high-performance liquid chromatography (HPLC) of cell wall mycolic acids, and genetic probes based on rRNA targets, e.g., AccuProbe, cannot discriminate among M. avium subspecies (2, 9). Given the differences in pathogenicity among M. avium subspecies and the implications regarding the infection source, a practical and accurate method of simply identifying M. avium subspecies is needed (13, 25, 35). In this study, we describe the specificity, discrimination capacity, and sensitivity of a novel five-target PCR, called the MAC multiplex, using a wide array of reference and clinical MAC isolates and numerous nonmycobacterial organisms.     

Genome‐wide transcriptome analyses reveal p53 inactivation mediated loss of miR‐34a expression in malignant peripheral nerve sheath tumours

Malignant peripheral nerve sheath tumours (MPNSTs) are aggressive soft tissue tumours that occur either sporadically or in patients with neurofibromatosis type 1. The malignant transformation of the benign neurofibroma to MPNST is incompletely understood at the molecular level. We have determined the gene expression signature for benign and malignant PNSTs and found that the major trend in malignant transformation from neurofibroma to MPNST consists of the loss of expression of a large number of genes, rather than widespread increase in gene expression. Relatively few genes are expressed at higher levels in MPNSTs and these include genes involved in cell proliferation and genes implicated in tumour metastasis. In addition, a gene expression signature indicating p53 inactivation is seen in the majority of MPNSTs. Subsequent microRNA profiling of benign and malignant PNSTs indicated a relative down‐regulation of miR‐34a in most MPNSTs compared to neurofibromas. In vitro studies using the cell lines MPNST‐14 (NF1 mutant) and MPNST‐724 (from a non‐NF1 individual) show that exogenous expression of p53 or miR‐34a promotes apoptotic cell death. In addition, exogenous expression of p53 in MPNST cells induces miR‐34a and other miRNAs. Our data show that p53 inactivation and subsequent loss of expression of miR‐34a may significantly contribute to the MPNST development. Collectively, our findings suggest that deregulation of miRNAs has a potential role in the malignant transformation process in peripheral nerve sheath tumours. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Development and Validation of Patient-Reported Outcome Measures for Sleep Disturbance and Sleep-Related Impairments

Study Objectives:

To develop an archive of self-report questions assessing sleep disturbance and sleep-related impairments (SRI), to develop item banks from this archive, and to validate and calibrate the item banks using classic validation techniques and item response theory analyses in a sample of clinical and community participants.

Design:

Cross-sectional self-report study.

Setting:

Academic medical center and participant homes.

Participants:

One thousand nine hundred ninety-three adults recruited from an Internet polling sample and 259 adults recruited from medical, psychiatric, and sleep clinics.

Interventions:

None.

Measurements and Results:

This study was part of PROMIS (Patient-Reported Outcomes Information System), a National Institutes of Health Roadmap initiative. Self-report item banks were developed through an iterative process of literature searches, collecting and sorting items, expert content review, qualitative patient research, and pilot testing. Internal consistency, convergent validity, and exploratory and confirmatory factor analysis were examined in the resulting item banks. Factor analyses identified 2 preliminary item banks, sleep disturbance and SRI. Item response theory analyses and expert content review narrowed the item banks to 27 and 16 items, respectively. Validity of the item banks was supported by moderate to high correlations with existing scales and by significant differences in sleep disturbance and SRI scores between participants with and without sleep disorders.

Conclusions:

The PROMIS sleep disturbance and SRI item banks have excellent measurement properties and may prove to be useful for assessing general aspects of sleep and SRI with various groups of patients and interventions.

Citation:

Buysse DJ; Yu L; Moul DE; Germain A; Stover A; Dodds NE; Johnston KL; Shablesky-Cade MA; Pilkonis PA. Development and validation of patient-reported outcome measures for sleep disturbance and sleep-related impairments. SLEEP 2010;33(6):781-792.     

Combat experience and the acquired capability for suicide

Rising suicide rates are an increasing concern among military personnel. The interpersonal‐psychological theory of suicide proposes that three necessary factors are needed to die by suicide: feelings that one does not belong with other people, feelings that one is a burden on others or society, and an acquired capability to overcome the fear and pain associated with suicide. The current study tests the theory's proposal that acquired capability may be particularly influenced by military experience, because combat exposure may cause habituation to fear of painful experiences such as suicide. Utilizing clinical and nonclinical samples of military personnel deployed to Iraq, results of the current study indicate that a greater range of combat experiences predicts acquired capability above and beyond depression and post‐traumatic stress disorder symptoms, previous suicidality, and other common risk factors for suicide. Combat experiences did not, however, predict perceived burdensomeness or thwarted belongingness. The authors discuss how combat experiences might serve as a mechanism for elevating suicide risk and implications for clinical interventions and suicide prevention efforts. © 2010 Wiley Periodicals, Inc. J Clin Psychol: 66:1–13, 2010.     

Croton cajucara crude extract and isolated terpenes: activity on Trypanosoma cruzi

Croton cajucara is a plant found in the Amazon region and is known for its medicinal properties. The effects of the methanolic extract of the stem bark of C. cajucara (MCC) and of the isolated terpenes, trans-dehydrocrotonin (t-DCTN) and acetyl aleuritolic acid (AAA), were investigated using four isolates of Trypanosoma cruzi. In assays with trypomastigotes, the extract was more active than the isolated compounds, presenting IC₅₀ in the range of 10 to 50 μg/mL. The trypanocidal effect of MCC, AAA and benznidazole was significantly higher in the GLT291 and C45 strains, which were recently isolated from wild animals. MCC and AAA caused a dose-dependent inhibition of epimastigote proliferation. In assays using intracellular amastigotes, AAA and MCC reduced the percent of infection and the endocytic index after 96 h of treatment, at concentrations that were non-toxic to the host cells. MCC inhibited the trypanothione reductase pathway in both epimastigotes and trypomastigotes of all the subpopulations. The absence of AAA activity on the trypanothione reductase pathway in epimastigotes of Dm28c suggests heterogeneity of the biochemical profile between this clone and the three strains. Epimastigotes and trypomastigotes (GLT291) were treated for 24 h with MCC or AAA, and both induced alterations of the plasma membrane, while AAA-treated epimastigotes also displayed mitochondrial damage.     

Steady-state visual evoked potentials reveal frontally-mediated working memory activity in humans

Steady-state visual evoked potentials (SSVEPs) reflect power changes at the stimulus driving frequency and have been used to assess brain activity reflecting cognitive processing. Only one study has demonstrated SSVEP modulation associated with working memory (WM), and none have compared the spatial localization of SSVEP modulations during WM performance with other brain imaging methods. Here we examined WM-related activity recorded with dense-array SSVEPs, analyzed using low resolution electromagnetic tomography, and compared the results to our previous findings using functional magnetic resonance imaging (fMRI). WM was associated with increased SSVEP activity over the right dorsolateral prefrontal cortex, paralleling our previous fMRI findings. Frontal WM-related SSVEP power correlated selectively with task performance. These results demonstrate the utility of SSVEPs for studying representational aspects of cognition.