Isolation of Mycobacterium kumamotonense from a patient with pulmonary infection and latent tuberculosis.

Mycobacterium kumamotonense is a novel, slow-growing non-chromogenic nontuberculous mycobacterium, which belongs to Mycobacterium terrae complex. We report, for the first time in Greece, the isolation of M. kumamotonense from an immunocompetent patient with pulmonary infection and latent tuberculosis. M. kumamotonense was identified by sequencing analysis of 16S rDNA and 65-kDa heat shock protein genes while by commercial molecular assays it was misidentified as Mycobacterium celatum. Antibiotic susceptibility testing was performed by the reference broth microdilution method. The strain was susceptible to amikacin, clarithromycin, rifampin, ciprofloxacin, moxifloxacin, rifabutin, ethambutol and linezolid.

Mycobacterium heraklionense sp. nov.: A case series.

Mycobacterium heraklionense sp. nov. (M. heraklionense) is a novel non-tuberculous mycobacterium belonging to the Mycobacterium terrae complex that has recently been described. It has a world-wide distribution. Recently, a case of tenosynovitis in an immunocompetent individual caused by M. heraklionense was reported, indicating that it has the ability to cause diseases. In the present study, in order to provide a more detailed profile of this mycobacterium and to obtain a more complete overall picture of its clinical significance, we report all available data regarding the initial 12 cases of its isolation. Of the 12 patients, 5 (42%) eventually died within a period of 3 months following the isolation of the mycobacterium. However, any connection between the presence of M. heraklionense and these deaths could not be documented. These 5 patients were all males with a mean age of 74.6 years suffering from serious underlying diseases, which most probably were the cause of death. Additional data from possible new cases of M. heraklionense isolation are anticipated.

Mycobacterium iranicum sp. nov., a rapidly growing scotochromogenic species isolated from clinical specimens on three different continents.

The isolation and characterization of a novel, rapidly growing, scotochromogenic mycobacterial species is reported. Eight independent strains were isolated from clinical specimens from six different countries of the world, two in Iran, two in Italy and one in each of following countries: Greece, The Netherlands, Sweden and the USA. Interestingly, two of the strains were isolated from cerebrospinal fluid. The strains were characterized by rapid growth and presented orange-pigmented scotochromogenic colonies. DNA-based analysis revealed unique sequences in the four regions investigated: the 16S rRNA gene, the rRNA gene internal transcribed spacer 1 and the genes encoding the 65 kDa heat-shock protein and the beta-subunit of RNA polymerase. The phylogenetic analysis placed the strains among the rapidly growing mycobacteria, being most closely related to Mycobacterium gilvum. The genotypic and phenotypic data both strongly supported the inclusion of the strains investigated here as members of a novel species within the genus Mycobacterium; the name Mycobacterium iranicum sp. nov. is proposed to indicate the isolation in Iran of the first recognized strains. The type strain is M05(T) ( = DSM 45541(T) = CCUG 62053(T) = JCM 17461(T)).

Survey of 150 strains belonging to the Mycobacterium terrae complex and description of Mycobacterium engbaekii sp. nov., Mycobacterium heraklionense sp. nov. and Mycobacterium longobardum sp. nov.

A thorough phenotypic and genotypic analysis of 150 strains belonging to the Mycobacterium terrae complex resulted in the identification of a number of previously unreported sequevars (sqvs) within the species known to belong to the complex. For the species Mycobacterium arupense, three sqvs were detected in the 16S rRNA gene, six sqvs in the hsp65 gene and 15 sqvs in the rpoB gene; in Mycobacterium senuense two sqvs were present in each of the three genetic regions; in Mycobacterium kumamotonense four, two and nine sqvs were found, respectively, and in M. terrae three, four and six sqvs were found, respectively. The inappropriate inclusion of Mycobacterium triviale within the M. terrae complex was confirmed. The limited utility of biochemical tests and of mycolic acid analyses for the differentiation of the members of M. terrae complex was also confirmed. The survey allowed the recognition of three previously undescribed species that were characterized by unique sequences in the 16S rRNA, hsp65 and rpoB genes. Mycobacterium engbaekii sp. nov. (proposed previously 40 years ago but never validly published) was characterized by pink photochromogenic pigmentation and rapid growth; phylogenetically it was related to Mycobacterium hiberniae. The type strain of this species, of which eight strains were investigated, is ATCC 27353(T) ( = DSM 45694(T)). A cluster of 24 strains was the basis for the description of Mycobacterium heraklionense sp. nov., which has an intermediate growth rate and is unpigmented; nitrate reductase activity is typically strong. Closely related to M. arupense with respect to the 16S rRNA gene, M. heraklionense sp. nov. could be clearly differentiated from the latter species in the other genetic regions investigated. The type strain is NCTC 13432(T) ( = LMG 24735(T) = CECT 7509(T)). Mycobacterium longobardum sp. nov., represented in the study by seven strains, was characterized by a unique phylogenetic location within the M. terrae complex, clearly divergent from any other species. The type strain is DSM 45394(T) ( = CCUG 58460(T)).

Antimicrobial susceptibility of Gram-positive cocci isolated from patients with conjunctivitis and keratitis in Crete, Greece.

PURPOSE: To assess the in vitro susceptibility of Streptococcus pneumoniae, Staphylococcus aureus and coagulase-negative staphylococci (CoNS) ocular isolates to antibiotics, and identify changing trends in resistance over a 10-year period. METHODS: All isolates from ocular infections collected between 2000 and 2009 were prospectively tested against several antibiotics in vitro. S. pneumoniae isolates (n = 93) were tested against 20 and S. aureus (n = 120) and CoNS (n = 214) against 19 antibiotics. To identify changes in susceptibility patterns, we compared results from 2000-2004 with those from 2005-2009. We also compared the antibiotic susceptibilities against aminoglycosides and quinolones between methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates. RESULTS: All S. pneumoniae isolates were susceptible to quinolones, and 99% were susceptible to chloramphenicol. Regarding S. aureus, we noted a significant increase in resistance against penicillin in recent years (p = 0.016). Over 90% of S. aureus isolates were susceptible to quinolones and aminoglycosides. MRSA isolates were more resistant to ciprofloxacin and ofloxacin than MSSA isolates were (p = 0.016). Concerning CoNS, a significant increase in susceptibility to amikacin was noted in the second study period (p = 0.01). CONCLUSION: Quinolones remain an excellent treatment option for bacterial conjunctivitis and keratitis due to Gram-positive cocci in our region.

Clinical significance and antibiotic susceptibilities of nontuberculous mycobacteria from patients in Crete, Greece.

AIM: To describe the clinical significance and antibiotic susceptibilities of nontuberculous mycobacteria (NTM) isolated from patients in Crete, Greece between January 2000 and December 2009. PATIENTS & METHODS: NTM identification was performed using conventional bacteriological methods and confirmed by molecular characterization with commercially available assays. Rare and novel species were identified by sequencing of the 16SrRNA and of the hsp65 genes. Antibiotic susceptibility testing was performed by E-test. Rapidly growing (RGM) and slowly growing (SGM) NTM were tested against 14 antimicrobials, including nine common ones, except for Mycobacterium avium and Mycobacterium intracellulare (MAC) complex isolates that were tested only against azithromycin, clarithromycin, linezolid and moxifloxacin. RESULTS: During the study period, 290 positive samples for NTM were recovered from 207 patients. Among the positive samples, 150 were identified as SGM and 57 as RGM. Overall, 50 patients met American Thoracic Society criteria for disease due to NTM, 42 by SGM and eight by RGM. Risk factors in patients with NTM disease were underlying lung diseases, mainly chronic obstructive pulmonary disease and asthma, smoking, rheumatoid arthritis, AIDS, alcohol or drug abuse, malignancies and bronchiectasis. The most common disease-causing species were the MAC complex (n = 25) followed by Mycobacterium kansasii (n = 10). Amikacin was the most active drug for RGM with 100% susceptibility. Macrolides were very active against isolates of the MAC complex, while tigecycline had excellent activity in vitro against RGM. M. kansasii was the most susceptible NTM species in vitro. CONCLUSION: Our study is the first to describe the clinical significance, risk factors and susceptibility patterns of NTM isolates in a Greek population.

GenoType® MTBDRplus compared with conventional drug-susceptibility testing of Mycobacterium tuberculosis in a low-resistance locale.

AIM: To determine the diagnostic accuracy of Geno Type® MTBDRplus compared with conventional proportional method (PM) drug susceptibility testing (DST) for detecting drug resistance of Mycobacterium tuberculosis (MTB) clinical isolates derived from the University Hospital of Heraklion, Crete, Greece. METHODS: All MTB clinical isolates obtained between January 2000 and May 2009 were tested prospectively using the PM DST. All isolates were also tested either prospectively (strains isolated after January 2006; n = 140) or retrospectively (January 2000-December 2005; n = 81) using MTBDRplus. RESULTS: Among the 221 MTB isolates tested, 19 were resistant to isoniazid based on PM DST. The MTBDRplus assay identified 18 INH-resistant strains. Of these, 12 strains gave positive hybridization results with the mutation-specific probe katG Ser315Thr. The other six INH-resistant strains carried the mutation 15C→T in the inhA promoter region. One INH-sensitive strain, as determined by PM DST, had a katG gene mutation. The sensitivity, specificity, and positive and negative predictive values of MTBDRplus for INH were 89.5, 99.5, 94.7 and 99%, respectively. The single rifampicin-resistant strain that was also the only multidrug resistance isolate gave a positive hybridization result with the mutation-specific probe MUT1: D516V, which confers resistance to rifampicin. CONCLUSION: Despite the advantages of MTBDRplus (turn-around time of only 1 working day) compared with PM DST, the latter is still required in every case of a MTB clinical isolate, as the molecular assay does not detect 100% of drug resistance, especially INH resistance.

Mycobacterium europaeum sp. nov., a scotochromogenic species related to the Mycobacterium simiae complex.

Four strains isolated in the last 15 years were revealed to be identical in their 16S rRNA gene sequences to MCRO19, the sequence of which was deposited in GenBank in 1995. In a polyphasic analysis including phenotypic and genotypic features, the five strains (including MCRO19), which had been isolated in four European countries, turned out to represent a unique taxonomic entity. They are scotochromogenic slow growers and are genetically related to the group that included Mycobacterium simiae and 15 other species. The novel species Mycobacterium europaeum sp. nov. is proposed to accommodate these five strains. Strain FI-95228(T) ( = DSM 45397(T)  = CCUG 58464(T)) was chosen as the type strain. In addition, a thorough revision of the phenotypic and genotypic characters of the species related to M. simiae was conducted which leads us to suggest the denomination of the 'Mycobacterium simiae complex' for this group.

Evaluation of GenoType mycobacteria direct assay in comparison with Gen-Probe Mycobacterium tuberculosis amplified direct test and GenoType MTBDRplus for direct detection of Mycobacterium tuberculosis complex in clinical samples.

Three molecular assays were evaluated for the direct detection of Mycobacterium tuberculosis complex bacteria in 125 respiratory and 22 nonrespiratory samples. The overall sensitivities obtained were as follows: GenoType MTBDRplus, 97.9%; GenoType Mycobacteria Direct, 93.7%; Gen-Probe Mycobacterium tuberculosis Amplified Direct Test, 89.6%. The specificity of the assays used was 100%.

Report of 2 indigenous cases of leprosy from a European country: use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for identification of Mycobacterium leprae directly from a clinical sample.

In this article, we report on 2 indigenous cases of leprosy detected in a European country. We also report on the use of polymerase chain reaction-restriction fragment length polymorphism analysis of hsp65 gene for rapid identification of Mycobacterium leprae directly from the clinical sample.

Molecular diagnostic tools in mycobacteriology.

Although the diagnosis of mycobacteriosis and susceptibility testing are still primarily based on conventional methods (staining, culture, biochemical analysis, proportional method), a series of molecular assays are increasingly introduced and incorporated in the workflow of clinical mycobacteriology laboratories worldwide. These assays are rapid and offer high sensitivities and specificities. In the present review, we describe the molecular assays concerning the early detection of Mycobacteria in clinical specimens, the identification of mycobacterial species, the detection of drug resistance and the typing for epidemiological investigations.

Miliary tuberculosis with no pulmonary involvement in myelodysplastic syndromes: a curable, yet rarely diagnosed, disease: case report and review of the literature.

BACKGROUND: Although tuberculosis is not uncommon among patients with myelodysplastic syndrome (MDS), only a few reports of such patients suffering from miliary tuberculosis (MT) exist. MT often presents as a fever of unknown origin and it is a curable disease, yet fatal if left untreated. CASE PRESENTATION: We report a case of MT with no clinical or laboratory indications of pulmonary involvement in a patient with MDS, and review the relevant literature. Mycobacterium tuberculosis was isolated from the liquid culture of a bone marrow aspirate. CONCLUSION: Even if the initial diagnostic investigation for a fever of obscure etiology is negative, MT should not be excluded from the differential diagnosis list. Since it is a curable disease, persistent and vigorous diagnostic efforts are warranted. In suspected cases, mycobacterial blood cultures should be collected as soon as possible after hospital admission and early bone marrow aspirate with mycobacterial cultures is advocated.

Mycobacterial species diversity at a general hospital on the island of Crete: first detection of Mycobacterium lentiflavum in Greece.

The objective of the present study was to investigate the diversity of mycobacterial isolates in a general hospital in Crete, Greece. 48 positive Lowenstein-Jensen cultures over a 3-y period were analysed by means of AccuProbe and GenoType assays. Non-tuberculous mycobacteria (NTM) comprised the majority of the isolates (56.3%, 27/48) vs 33.3% (16/48) of M. tuberculosis; 10.4% of the isolates could not be classified. Among NTM, M. lentiflavum was the predominant species isolated (9/27) followed by M. kansasii, M. gordonae and M. peregrinum, whereas no M. avium complex isolates were detected. This is the first detection of M. lentiflavum in Greece. The susceptibilities of the M. lentiflavum isolates to an extended panel of antibiotics were determined by the proportions method and the medical files of the 9 patients were reviewed. Three isolates were from urine, which is an unusual site. All strains exhibited multidrug resistance. The patients were adults with immunosuppression or predisposing conditions for NTM infection. Diagnosis of true infection was either not pursued or the patients died shortly after isolation.

Genital tuberculosis in a tamoxifen-treated postmenopausal woman with breast cancer and bloody vaginal discharge.

BACKGROUND: Female genital tuberculosis is an uncommon disease that is rarely diagnosed in developed countries. CASE PRESENTATION: A 61-year-old postmenopausal woman who had undergone surgery and treated with adjuvant chemotherapy for infiltrating ductal carcinoma of the breast five years ago, presented with bloody vaginal discharge, fatigue, weight loss, and low grade fevers at night for two months. Histological examination of the endometrium, done based on the suspicion of a second primary cancer due to the tamoxifen therapy, revealed a granulomatous reaction. Liquid and solid mycobacterial cultures of the tissues were performed. Although the acid fast staining was negative, the liquid culture was positive for Mycobacterium tuberculosis. Involvement of other systems was not detected. The patient was treated with a three-drug antituberculosis regimen for 9 months and recovered fully. CONCLUSION: Female genital tuberculosis is a rare but curable disease that should be included in the differential diagnosis of women with menstrual problems. Early diagnosis is important and may prevent unnecessary invasive procedures for the patient.

Use of the GenoType Mycobacterium CM and AS assays to analyze 76 nontuberculous mycobacterial isolates from Greece.

Seventy-six nontuberculous mycobacterial isolates obtained from patients living in Greece were analyzed with the GenoType Mycobacterium CM (for common mycobacteria) and AS (for additional species) assays. GenoType correctly identified all but one of the mycobacterial species. For this species, additional probes should be designed and added to the strip.

Evaluation of the fully automated Bactec MGIT 960 system for the susceptibility testing of Mycobacterium tuberculosis to first-line drugs: a multicenter study.

The accuracy of the Bactec MGIT 960 system for susceptibility testing of 177 clinical isolates of Mycobacterium tuberculosis to first line drugs (isoniazid, rifampicin, ethambutol and streptomycin) was compared with the agar reference method. The sensitivity, the ability to detect resistance, of the MGIT system was 100%, while the specificity, the ability to detect susceptibility, ranged from 98.6% to 100% for all drugs tested.

Multicenter evaluation of the fully automated Bactec MGIT 960 system for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide: comparison with the radiometric Bactec 460TB system.

One hundred and fifty clinical isolates of Mycobacterium tuberculosis were tested for susceptibility to pyrazinamide using the fully automated Bactec MGIT 960 system and the radiometric Bactec 460TB system. The overall concordance rate between MGIT 960 and radiometric system was 100% and the mean turnaround times to report the susceptibility test results were almost identical (6.37 and 6.8 days, respectively).

Combined use of the fully automated Bactec MGIT 960 system and a PCR-restriction fragment length polymorphism analysis for routine detection and identification of mycobacteria from clinical samples.

The combined use of Bactec MGIT 960 system and a PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis was assessed for the rapid detection and identification of mycobacteria from clinical samples. The diagnostic sensitivity and the time of detection of MGIT 960 system were significantly higher than of Löwenstein-Jensen medium. PCR-RFLP identification analysis results were in concordance with those obtained by the conventional biochemical tests.