Clinicopathologic implications of MDM2, p53 and K-ras gene alterations in osteosarcomas: MDM2 amplification and p53 mutations found in progressive tumors

The role of rapidly growing mycobacteria in the pathogenesis of pulmonary disease is being increasingly recognized; however, the clinical significance of these mycobacteria in patients with underlying malignancy has not been well studied. Over a 6-year period, 37 cancer patients with rapidly growing mycobacteria isolated from respiratory specimens were identified at our center. Mycobacterium chelonae group was isolated in 24 cases and Mycobacterium fortuitum in 13 cases. Of the 24 cases with cultures yielding Mycobacterium chelonae group, eight met the study criteria for infection and were determined to be clinically significant, whereas only one of the Mycobacterium fortuitum isolates was determined to represent infection. An average of two antimicrobial agents were used for treatment, most commonly clarithromycin, ciprofloxacin, and trimethoprim/sulfamethoxazole. Although the isolation of rapidly growing mycobacteria represents colonization in most cases, these bacteria, especially the Mycobacterium chelonae group, may cause pulmonary disease in cancer patients. The clinical and radiological findings are usually non-specific in this population, and patients with respiratory cultures yielding rapidly growing mycobacteria should be assessed carefully to distinguish infection from colonization. Effective therapy can be provided with oral regimens that include at least two antibiotics to which the organism is susceptible.     

Failure to recognize rapidly growing mycobacteria in a proficiency testing sample without specific request--a wider diagnostic problem?

Fifty participants in the Swiss External Quality Control Program in Bacteriology/Mycology received a diagnostic sample containing Mycobacterium fortuitum. Only 31 used some sort of acid-fast stains, and 13 reported the diagnosis of M. fortuitum or rapidly growing mycobacteria. We conclude that the presence of 'rapid growers' in routine bacteriology samples is underestimated, and that acid-fast stains should be performed on suspicious Gram-positive rods.     

MR imaging of carcinoma within urinary bladder diverticulum

Mycobacterium fortuitum is a rapidly growing atypical mycobacterium frequently reported as a postsurgical wound complication from a major surgical procedure. We present a unique case of M. fortuitum infection occurring in a 4-year-old boy after a minor punch biopsy surgical procedure. As far as we know there has been no published case of M. fortuitum occurring after a punch biopsy procedure.     

Studies on diurnal fluctuation and variation among days in the relative frequencies of the stages of the cycle of seminiferous epithelium in the mouse (author''s transl)]

The clinical records of 7 patients referred to the National Jewish Hospital and Research Center over a 6-year period for evaluation of an abnormal chest x-ray and repeated sputum isolates of rapidly growing mycobacteria (Runyon's Group IV) were reviewed to determine the potential pathogenicity of these organisms. Mycobacterium fortuitum was isolated from 5 patients and Mycobacterium chelonei from 2. Haemoptysis, cough and weight loss were prominent in 6. Three had rheumatoid arthritis. Although two demonstrated cutaneous anergy, lymphocyte responsiveness to PHA was normal. PPD-F was not useful in skin testing or in the in vitro evaluation of lymphocyte function. Histologic examination of the lungs of 2 patients demonstrated caseating granulomata. One patient died of massive pulmonary haemorrhage soon after intiation of therapy. Multi-drug treatment regimens generally resulted in progressive sterilization of the sutum and improvement in the appearance of the chest x-ray. We conclude that some rapidly growing mycobacteria can cause potentially fatal cavitary lung disease and that intensive anti-tuberculosis therapy may successfully alter its course.     

katGI and katGII encode two different catalases-peroxidases in Mycobacterium fortuitum

It has been suggested that catalase-peroxidase plays an important role in several aspects of mycobacterial metabolism and is a virulence factor in the main pathogenic mycobacteria. In this investigation, we studied genes encoding for this protein in the fast-growing opportunistic pathogen Mycobacterium fortuitum. Nucleotide sequences of two different catalase-peroxidase genes (katGI and katGII) of M. fortuitum are described. They show only 64% homology at the nucleotide level and 55% identity at the amino acid level, and they are more similar to catalases-peroxidases from different bacteria, including mycobacteria, than to each other. Both proteins were found to be expressed in actively growing M. fortuitum, and both could also be expressed when transformed into Escherichia coli and M. aurum. We detected the presence of a copy of IS6100 in the neighboring region of a katG gene in the M. fortuitum strain in which this element was identified (strain FC1). The influence of each katG gene on isoniazid (isonicotinic acid hydrazide; INH) susceptibility of mycobacteria was checked by using the INH-sensitive M. aurum as the host. Resistance to INH was induced when katGI was transformed into INH-sensitive M. aurum, suggesting that this enzyme contributes to the natural resistance of M. fortuitum to the drug. This is the first report showing two different genes encoding same enzyme activity which are actively expressed within the same mycobacterial strain.     

Mutation in the signal-transducing chain of the interferon-gamma receptor and susceptibility to mycobacterial infection

IFN-gamma is critical in the immune response to mycobacterial infections, and deficits in IFN-gamma production and response have been associated with disseminated nontuberculous mycobacterial infections. Mutations in the IFN-gamma receptor ligand-binding chain (IFNgammaR1) have been shown to confer susceptibility to severe infection with nontuberculous mycobacteria. However, mutations in the IFN-gamma receptor signal-transducing chain (IFNgammaR2) have not been described. We describe a child with disseminated Mycobacterium fortuitum and M. avium complex infections and absent IFN-gamma signaling due to a mutation in the extracellular domain of IFNgammaR2. In vitro cytokine production by patient PBMCs showed 75% less PHA-induced IFN-gamma production than in normal cells, while patient PHA-induced TNF-alpha production was normal. The normal augmentation of TNF-alpha production when IFN-gamma was added to endotoxin was absent from patient cells. Expression of IFNgammaR1 was normal, but there was no phosphorylation of Stat1 in response to IFN-gamma stimulation. DNA sequence analysis of the gene for IFNgammaR2 showed a homozygous dinucleotide deletion at nucleotides 278 and 279, resulting in a premature stop codon in the protein extracellular domain. This novel gene defect associated with disseminated nontuberculous mycobacterial infection emphasizes the critical role that IFN-gamma plays in host defense against mycobacteria.     

Clinical significance of the isolation of rapidly growing mycobacteria

BACKGROUND: To assess the clinical significance of the isolates of rapid-growing mycobacteria in a Universitary hospital from Madrid (Spain). PATIENTS AND METHODS: Review of medical records from patients with isolates of rapid-growing mycobacteria identified between 1979 and 1996 in the Microbiology department of the Fundación Jiménèz Díaz (Madrid, Spain). RESULTS: Rapid-growing mycobacteria were isolated from 28 patients during the study period (13 M. chelonae, 10 M. fortuitum, 2 M. mucogenicum, 1 M. marinum, 1 M. smegmatis and 1 M. flavascens). Clinical records of 26 patients were reviewed, being the isolate significative in 10 cases (5 soft tissue infections, 2 peritonitis in patients undergoing Continuous Ambulatory Peritoneal Dialysis [CAPD], 1 urinary tract infection, 1 osteomyelitis and 1 catheter-related soft-tissue infection). No patient was HIV+. All infections cured except 2 of them (the urinary tract infection and the osteomyelitis). Catheter withdrawal was needed in 3 cases (peritonitis in CAPD and catheter-related soft-tissue infection), apart from proper antimicrobial therapy. CONCLUSION: The most frequent rapid-growing mycobacteria isolated were those of the M. fortuitum complex. In our experience, isolation of rapid-growing mycobacteria from skin and soft-tissue samples was usually clinically significant, while isolates from respiratory tract, gut and blood cultures are always nonsignificant.     

Mediastinitis due to Mycobacterium fortuitum infection following Fontan operation in a child

Here we report a case of mediastinitis due to Mycobacterium fortuitum infection in a child after a Fontan operation. To our knowledge this is the first report of atypical mycobacterial mediastinal infection after congenital heart surgery. Atypical mycobacteria can be the cause of "culture negative" sternal and thoracotomy wound infections. A brief review of the literature is included in the discussion.     

Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis

An assay is described utilizing PCR technology for a rapid diagnostic test to identify fish infection with Mycobacterium marinum, M. fortuitum and M. chelonae. A 924 bp DNA fragment from a highly conserved area of the mycobacterial 16S rRNA gene was amplified using mycobacteria genus-specific primers and digested with restriction enzymes (BanI and ApaI). This examination yielded unique restriction patterns for each mycobacterial specie enabling identification of mycobacteria infecting fish to the species level. The protocol can be applied to purified DNA, a simple colony preparation or infected fish tissue. This protocol can be completed in 1-2 days.     

Mycobacterium fortuitum mastoiditis

Improved techniques of bacteriologic identification have led to increasing recognition of the clinical significance of the atypical or anomymous mycobacteria. Mycobacterium fortuitum, included in group IV of Runyon's classification because of its characteristic rapid growth, is widespread in nature as a saprophyte. Its facultative pathogenicity has received increasing attention in the literature recently with reports of a number of isolated infections, epidemics, and deaths. We report a case of mastoiditis due to M fortuitum and discuss the pathogenesis and treatment. This is the first known report of the organism causing otolaryngologic disease.     

Bacteriology of mycobacteria: taxonomic and morphological characteristics

Bacteriological characteristics of organisms belonging to Genus Mycobacterium which involves more than 60 species are described. Mycobacterial organisms can be divided into the following groups having differential characteristics, on the basis of the results of biological, biochemical, and genetic investigations, including lipid analysis, DNA probe test, and comparative 16S ribosomal RNA sequencing. First, Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, etc.). Second, cultivable but slowly growing nontuberculous mycobacteria, including photochromogens (Runyon Group I) such as M. kansasii, M. marinum, M. simiae, M. intermedium, and M. asiaticum, scotochromogens (Runyon Group II) such as M. scrofulaceum, M. szulgai, M. injectum, M. lentiflavum, and M. gordonae, nonphotochromogenens (Runyon Group III) such as M. avium, M. intracellulare, M. xenopi, M. malmoense, M. genavense, M. celatum, and M. gastri. Third, cultivable rapidly growing nontuberculous mycobacteria (Runyon Group IV) including M. fortuitum, M. chenolae, M. abscessus, M. phlei, and M. smegmatis. Fourth, noncultivable mycobacteria including M. leprae. About 30 species of Mycobacterium cause pulmonary, dermal, lymphatic, and disseminated infections in human beings. This paper mainly deals with the taxonomic, morphological, and other biological characteristics of these mycobacterial organisms.     

Infections due to nontuberculous mycobacteria in children with leukemia

We reviewed the spectrum of infections due to nontuberculous mycobacteria (NTM) in children with leukemia. Three children acquired such infections. One patient developed pneumonia after the cessation of chemotherapy when Mycobacterium xenopi was identified in his lung biopsy specimen. He required 2 years of treatment with antituberculous agents and clarithromycin. Cultures of central and peripheral blood specimens from two patients yielded Mycobacterium fortuitum and Mycobacterium chelonae, respectively. Broviac catheters were likely the source of infection. Removal of the catheters and antibiotic treatment resulted in cure. Central venous catheters in leukemic children are potential sources of infection. For febrile neutropenic children with leukemia who do not respond to antibiotic therapy, cultures positive for diphtheroids or negative routine bacterial and fungal cultures should raise a suspicion for infections due to NTM. Systemic infections may require up to 2 years of therapy. Removal of the infected catheters during persistent or recurrent infections in necessary for control of the infection.     

Nontuberculous mycobacterial infection of the central nervous system in patients with AIDS

Infections due to nontuberculous mycobacteria (NTM) are especially common in patients with AIDS. Meningitis due to NTM, however, is rare. A search for CSF cultures positive for NTM over the past 11 years at our hospital yielded 16 cases. Of these, 15 were caused by Mycobacterium avium-intracellular (MAI), and one was caused by M fortuitum. All patients with MAI infection had widespread dissemination and at least one risk factor for AIDS. Clinical features included weight loss, altered mentation, and seizures. Analysis of cerebrospinal fluid revealed a mildly elevated leukocyte count with lymphocyte predominance and normal protein and glucose values. All direct smears were negative for acid-fast bacilli. In-hospital mortality was 67%. The patient with infection due to M fortuitum had a preexisting diagnosis of AIDS and had a right upper lobe pneumonia and headaches. Cranial CT showed an enlarged infundibulum of the pituitary gland. Results of CSF analysis were essentially normal, and direct smears were negative. He left the hospital against medical advice. Our study indicates that the finding of MAI in the CSF in patients with AIDS is associated with an in-house mortality of 67% indicating a very poor prognosis.     

Mycobacterium novocastrense sp. nov., a rapidly growing photochromogenic mycobacterium

A strain isolated from a biopsy sample taken from a slowly spreading skin granulation on a child's hand was found to have properties consistent with its classification in the genus Mycobacterium. An almost complete gene sequence of the 16S rRNA of the strain was determined following the cloning and sequencing of the amplified gene. The sequence was aligned with those available for mycobacteria, and phylogenetic trees were inferred with four tree-making algorithms. The organism, which formed a distinct phyletic line within the evolutionary radiation occupied by rapidly growing mycobacteria, was readily distinguished from members of validly described species of rapidly growing mycobacteria on the basis of its mycolic acid pattern and a number of other phenotypic features, notably its ability to form yellow pigmented colonies when incubated in the light. The name proposed for this new species is Mycobacterium novocastrense. The type strain is DSM 44203.     

Severe gastrointestinal hemorrhage due to Mycobacterium avium complex in a patient receiving immunosuppressive therapy

Intense immunosuppressive therapy is used frequently for treatment of systemic vasculitides, collagenoses, rapidly progressive glomerulonephritis, and after organ transplantation. Numerous serious treatment-related side effects include localized or disseminated opportunistic infections, and require careful monitoring of immunosuppressed patients. Gastrointestinal infections with Mycobacterium avium complex (MAC) or other nontuberculous mycobacteria have been previously identified in HIV seropositive patients only. We now report the first case of an HIV seronegative patient who received immunosuppressive therapy for rapidly progressive glomerulonephritis. The patient presented with severe lower gastrointestinal bleeding and was diagnosed to have ulcerative colitis due to infection with MAC. The patient recovered promptly after administration of antimycobacterial therapy. MAC infection should be included in the differential diagnosis of gastrointestinal bleeding in all immunodeficient patients. The significance of repeated colonoscopy to obtain multiple biopsy specimens with histological examination for foam cells and specific staining for acid-fast organisms is emphasized.     

A cis-regulatory element within the 5'' flanking region of arylsulfatase gene of sea urchin, Hemicentrotus pulcherrimus

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.     

Epidemiology of disease caused by nontuberculous mycobacteria

The clinically important nontuberculous mycobacteria include M. kansasii, M. genavense, M. marinum, M. simiae, M. scrofulaceum and M. szulgai, M. avium, M. haemophilum, M. intracellulare, M. malmoense, M. ulcerans, and M. xenopi, M. abscessus, M. chelonae, M. fortuitum, and (rarely) M. smegmatis. Four clinical syndromes account for nearly all cases: pulmonary disease, lymphadenitis, skin or soft tissue disease, and disseminated disease in AIDS. M. avium and M. intracellulare (known together as M. avium complex), are the most common causes of pulmonary disease, lymphadenitis, and disseminated disease. All four clinical syndromes seem to be increasing in frequency, particularly in immunosuppressed hosts. Nontuberculous mycobacteria are acquired from the environment, but specific reservoirs of these organisms leading to human disease have not been defined.     

Ultrastructural studies in epidermolysis bullosa hereditaria. III. Recessive dystrophic types with dermolytic blistering (Hallopeau-Siemens types and inverse type)

In this paper the authors describe and analyse results that they obtained by infection of the guinea pig organism carried out by subcutaneous or intratracheal application with five mycobacterial strains, namely Myco bovis BCG-Praha, Myco the H37Ra, Myco Kansasii, Myco fortuitum and Myco smegmatis. At predetermined time intervals following subcutaneously or intratracheally performed infection (on 7th, 16th, 28th and 56th day after infection) transplantation of a Deals' guinea pig sarcoma cell suspension was carried out in guinea pigs by the intraltracheal route. As it appears from the results gained the applied mycobacteria exhibit a partial inhibition of growth od Deals' guinea pig sarcoma cells of different character. From among the utilized strains the Myco bovis BCP-Praha and the Myco tbc H37Ra exhibited the highest, Myco fortuitum and Myco smegmatis the lowest inhibitory activity. Intratracheally performed infections yielded in general better results on the growth inhibition than infections carried out with the same strain but by the subcutaneous route. Furthermore, in the experiments reported on in the present paper the authors could verify their earlier experience, namely that inhibition of growth of sarcoma cells is most pronounced at the time of maximal biological activity (logarithmic phase of multiplication) of the applied mycobacterium.     

Variables affecting results of sodium chloride tolerance test for identification of rapidly growing mycobacteria

The sodium chloride tolerance test is often used in the identification of rapidly growing mycobacteria, particularly for distinguishing between Mycobacterium abscessus and Mycobacterium chelonae. This test, however, is frequently unreliable for the identification of some species. In this study we examined the following variables: medium manufacturer, inoculum concentration, and atmosphere and temperature of incubation. Results show that reliability is improved if the test and control slants are inoculated with an organism suspension spectrophotometrically equal to a 1 McFarland standard. Slants should be incubated at 35 degrees C in ambient air and checked weekly for 4 weeks. Growth on control slants should be critically evaluated to determine the adequacy of the inoculum; colonies should number greater than 50. Salt-containing media should be examined carefully to detect pinpoint or tiny colonies, and colonies should number greater than 50 for a positive reaction. Concurrent use of a citrate slant may be helpful for distinguishing between M. abscessus and M. chelonae. Molecular methodologies are probably the most reliable means for the identification of rapidly growing mycobacteria and should be used, if possible, when unequivocal species identification is of particular importance.     

Phase transitions in phospholipid monolayers at the air-water interface: a fluorescence study

A study was made of the specific sensitivity to tuberculin in the animals vaccinated with BCG in case of their additional sensitization with various atypical mycobacteria. The mentioned experimental study appeared to be necessary for the purpose of a more proper treatment of the epidemiological date referred to the sensitivity of man to tuberculin and sensitins against the background of mass BCG vaccination. Preliminary BCG vaccination of the animals with their subsequent infection with atypical mycobacteria altered the allergic response to the antigens from the mycobacteria increasing the response reactions not only to tuberculin, but also to sensitins from mycobacteria of the I--III groups by Runyon's classification, closely connected in antigenic respect with mycobacteria tuberculosis. Skin reactions to sensitins from the saprophytic mycobacteria which had in their composition much less common antigens with mycobacteria tuberculosis, remained at the low level. Sensitization with atypical mycobacteria of animals preliminarily vaccinated with BCG failed to cause significant influence on the production of immunity to the subsequent virulent infection with tuberculosis.