First case of Mycobacterium marseillense lymphadenitis in a child.

BACKGROUND: Nontuberculous mycobacteria (NTM) are pathogens that commonly affect the paediatric population and its most frequent manifestation is a cervicofacial lymphadenopathy. With the improvement of technologies, new species have been recently identified. CASE PRESENTATION: We report the first case of NMT lymphadenitis in a child caused by Mycobacterium marseillense, a newly described species belonging to Mycobacterium avium complex. CONCLUSIONS: Improving the identification of these newly discovered mycobacteria, further information will be available about their clinical involvement and their best treatment.

Non-tuberculous mycobacteria: epidemiological pattern in a reference laboratory and risk factors associated with pulmonary disease.

The diseases caused by non-tuberculous mycobacteria (NTM), in both AIDS and non-AIDS populations, are increasingly recognized worldwide. Although the American Thoracic Society published the guidelines for diagnosis of NTM pulmonary disease (NTM-PD), the diagnosis is still difficult. In the first part of the study, we collected data on NTM isolates in the Mycobacteriology Laboratory of Careggi Hospital (Florence, Italy) and analysed the epidemiological data of NTM isolates. Then, to analyse the risk factors associated to NTM-PD, we studied the presence of ATS/IDSA criteria for NTM-PD in patients who had at least one positive respiratory sample for NTM and were admitted to the Infectious Disease Unit and the Section of Respiratory Medicine. We selected 88 patients with available full clinical data and, according to ATS/IDSA criteria, classified 15 patients (17%) as NTM-PD cases and 73 as colonized patients (83%). When comparing colonized and NTM-PD patients we did not find significant differences of age, gender and comorbidity. We observed that Mycobacterium avium and M. intracellulare were statistically associated with NTM-PD (P = 0·001) whereas M. xenopi was statistically associated with colonization. Although the number of studied patients is limited, our study did not identify risk factors for NTM-PD that could help clinicians to discriminate between colonization and disease. We underline the need of close monitoring of NTM-infected patients until the diagnosis is reasonably excluded. Further larger prospective studies and new biological markers are needed to identify new useful tools for the diagnosis of NTM-PD.

Use of BACTEC MGIT 960 for recovery of mycobacteria from clinical specimens: multicenter study.

The BACTEC MGIT 960 instrument is a fully automated system that exploits the fluorescence of an oxygen sensor to detect growth of mycobacteria in culture. Its performance was compared to those of the radiometric BACTEC 460 instrument and egg-based Lowenstein-Jensen medium. An identical volume of sample was inoculated in different media, and incubation was carried out for 6 weeks with the automatic systems and for 8 weeks on solid media. A total of 2,567 specimens obtained from 1,631 patients were cultured in parallel. Mycobacteria belonging to nine different taxa were isolated by at least one of the culture systems, with 75% of them being represented by Mycobacterium tuberculosis complex. The best yield was obtained with the BACTEC 460 system, with 201 isolates, in comparison with 190 isolates with the BACTEC MGIT 960 system and 168 isolates with Lowenstein-Jensen medium. A similar but not significant difference was obtained when the most-represented organisms, the M. tuberculosis complex, Mycobacterium xenopi, and the Mycobacterium avium complex, were analyzed separately and when combinations of a solid medium with the BACTEC MGIT 960 system and with the BACTEC 460 system were considered. The shortest times to detection were obtained with the BACTEC MGIT 960 system (13.3 days); 1.5 days earlier than that with the BACTEC 460 system (14.8 days) and 12 days earlier than that with Lowenstein-Jensen medium (25.6 days). The BACTEC MGIT 960 system had a contamination rate of 10.0%, intermediate between those of the radiometric system (3.7%) and the egg-based medium (17.0%). We conclude, therefore, that the BACTEC MGIT 960 system is a fully automated, nonradiometric instrument that is suitable for the detection of growth of tuberculous and other mycobacterial species and that is characterized by detection times that are even shorter than that of the "gold standard," the BACTEC 460 system. The contamination rate was higher than that for the radiometric BACTEC 460 system and needs to be improved.

HPLC does not differentiate Mycobacterium paratuberculosis from Mycobacterium avium.

HPLC, which is gaining its place as identification tool in mycobacteriology laboratories, has been proposed to distinguish Mycobacterium paratuberculosis from Mycobacterium avium. We had reported no significant difference between M. avium and M. paratuberculosis reference strain ATCC 19698. Because of the advantages offered by such a method, we enlarged our observations to include more isolates of M. paratuberculosis. Within the double cluster of peaks obtained by both M. avium and M. paratuberculosis, we could not find a consistent difference typical of M. paratuberculosis. Therefore, the present study confirmed that M. avium and M. paratuberculosis could not be distinguished by HPLC, raising doubts of a straightforward use of HPLC to identify M. paratuberculosis.

Early detection of Mycobacterium tuberculosis in BACTEC cultures by ligase chain reaction.

The LCx Mycobacterium tuberculosis ligase chain reaction system (Abbott Diagnostic Division, Abbott Park, Ill.) was used to detect M. tuberculosis in 150 consecutive BACTEC vials on the day on which a positive growth index (GI) was recorded. By LCx, M. tuberculosis DNA was detected in BACTEC vials on average 2.6 days before the presence of acid-fast bacilli could be confirmed by microscopic examination. A total of 106 of 108 M. tuberculosis isolates were detected without centrifugation from bottles presenting very low GIs (average, 70; median, 33). No false-positive result was obtained from nontuberculous mycobacteria or from isolates with contaminants.

Mycobacterium genavense in AIDS patients, report of 24 cases in Italy and review of the literature.

Mycobacterium genavense is a frequently missed agent of disseminated disease in AIDS patients. The increasing frequency with which such organism is being isolated in Italy suggested a comparison of local survey with data reported in literature. Isolates presumed to belong to the species M. genavense were centralized and identified by means of genomic sequencing and/or HPLC analysis of cell wall mycolic acids; clinical data were obtained from relevant patients' record and collected using a proper questionnaire. In 24 cases in which this organism has been isolated in Italy M. genavense was grown, prevalently from blood, in liquid medium after an average of six weeks of incubation. In overwhelming majority, patients were males, presented other opportunistic diseases and were characterized by very low CD4+ counts (average 23/microl); most frequent symptoms were fever, anemia and weight loss. All but two patients, who died before the mycobacterial infection was diagnosed, were treated with at least three drugs; the mean survival was close to one year. A review of literature reports revealed a wide overlapping of clinical and microbiological features.

Pulmonary infection due to Mycobacterium szulgai, case report and review of the literature.

We describe the case of a patient with a chronic pulmonary infection due to a mycobacterium tentatively identified as Mycobacterium flavescens, but finally shown to be Mycobacterium szulgai; this is the first M. szulgai infection reported in Italy. The patient responded to treatment with multiple antituberculosis drugs only after two cycles of 10 and 6 months, respectively. The literature concerning previous case reports in which M. szulgai is involved is revised and the difficulty concerning the identification of this rare mycobacterium, along with its in vitro and in vivo susceptibility, are discussed.

Multicenter comparison of ESP Culture System II with BACTEC 460TB and with Lowenstein-Jensen medium for recovery of mycobacteria from different clinical specimens, including blood.

The recently developed ESP Culture System II (AccuMed, Chicago, Ill.) was compared with radiometric BACTEC 460TB (Becton Dickinson, Towson, Md.) and with Lowenstein-Jensen medium for recovery of mycobacteria from over 2,500 clinical specimens both of respiratory and nonrespiratory origin, including blood. The majority of the 219 mycobacterial isolates (129) belonged to the Mycobacterium tuberculosis complex, followed by 37 isolates of the Mycobacterium avium complex (MAC) and 53 isolates of eight other mycobacterial species. Rates of recovery obtained with BACTEC, ESP, and Lowenstein-Jensen medium were 89, 79, and 64%, respectively, with such differences being statistically significant. Different media and systems appeared to behave differently when the more frequently detected organisms were considered: M. tuberculosis complex isolates grew better with BACTEC, and MAC isolates grew better with ESP. An analysis of the combinations of Lowenstein-Jensen medium with BACTEC and with ESP did not reveal significant differences in recovery rates. With regard to the times needed for the detection of positive cultures, they were significantly longer on Lowenstein-Jensen medium (average, 28 days) than with the remaining two systems, between which there was no difference (average, 18 days). We conclude, therefore, that the ESP system, when used in combination with a solid medium, performs as well as the thoroughly validated radiometric BACTEC system and offers the advantages of full automation and absence of radioisotopes.

Evaluation of a commercial ligase chain reaction kit (Abbott LCx) for direct detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens.

Direct detection of Mycobacterium tuberculosis by means of a commercial ligase chain reaction DNA amplification method (LCx M. tuberculosis; Abbott Diagnostics Division, Abbott Park, Ill.) was investigated with 511 (including 147 extrarespiratory) specimens collected from 358 patients. LCx results were compared with standard microbiological data, and conflicting cases were resolved according to the final clinical diagnosis. M. tuberculosis was detected in 45 of 358 subjects by means of the LCx test. The test was negative for all 30 specimens with mycobacteria other than M. tuberculosis. The sensitivity, specificity, and positive and negative predictive values for the LCx test, compared with culture results, were 93.90, 92.31, 70.00, and 98.75%, respectively; these values rose in resolved cases to 95.53, 99.25, 97.27, and 98.75%, respectively. With respiratory specimens, for which the LCx system is licensed, the sensitivity reached 98.97%. In patients with a final clinical diagnosis of tuberculosis the sensitivity of the LCx system was 89.36% compared to 82.98% for cultures and 78.72% for microscopy. We conclude that the LCx test is user friendly, rapid, fairly sensitive, and highly specific. It can also be effectively used on extrapulmonary specimens provided possible false-negative results are taken into account. However, the use of LCx test appears to be less appropriate for the monitoring of antituberculosis therapy, as the majority of samples from treated tuberculosis patients gave consistently positive results, despite the sterilization of cultures.

Mycobacterium malmoense in Italy: the modern Norman invasion?

The isolation of Mycobacterium malmoense has for a long time been restricted to few countries of Northern Europe; reports from countries other than Sweden, Great Britain and Finland are rare and the first Italian case report has been published in 1995. Since 1988, however, fifteen strains of M. malmoense have been isolated in Italy, eleven of which in the last two years; of these, ten appeared clinically significant on the basis of medical records. The susceptibility of the strains and the role of high performance liquid chromatography of cell wall mycolic acids for a reliable identification are discussed.

Cervical lymphadenitis due to an unusual mycobacterium.

A scotochromogenic acid-fast bacillus was isolated from a lymph node of a 2-year-old female. On the basis of conventional testing, the mycobacterium appeared to be Mycobacterium scrofulaceum. Its mycolic acid profile, however, was not identical to that of Mycobacterium scrofulaceum but was similar to that of Mycobacterium interjectum. Direct sequencing of the 16S rRNA gene revealed a unique nucleic acid sequence, suggesting that the isolate represents a previously undescribed pathogenic species.

Characterization of mycobacterial isolates phylogenetically related to, but different from Mycobacterium simiae.

The use of high-performance liquid chromatography (HPLC) revealed four previously unreported profiles within a group of mycobacteria consisting of 14 clinical isolates. These mycobacteria, whose identification by conventional tests appeared problematic, mostly resembled Mycobacterium avium complex or Mycobacterium simiae. Genetic analysis revealed, within this group, six different nucleic acid sequences in a hypervariable 16S rRNA segment, but all the isolates appeared to be phylogenetically related to M. simiae. Six isolates representing the largest of groups defined by means of genetic sequencing turned out to belong to the newly described species Mycobacterium lentiflavum. Furthermore, three such clusters precisely coincided with three of those defined by HPLC, while the three remaining clusters shared almost identical HPLC profiles. All but one strain (which, although clearly not belonging to the M. avium complex, hybridized with specific commercial DNA probes) showed high-grade resistance to the majority of antimycobacterial drugs. Three of the isolates were clinically significant according to stringent criteria. Sophisticated techniques, like genetic sequencing or HPLC, by now seem indispensable for differentiating unusual and new mycobacteria from well-established ones.

Evaluation of reformulated chemiluminescent DNA probe (AccuProbe) for culture identification of Mycobacterium kansasii.

A panel of 104 isolates belonging to the species Mycobacterium kansasii and 78 mycobacterial isolates belonging to other species was tested in parallel with the present commercially available DNA probe (AccuProbe; Gen-Probe) and with a new probe just developed by the same manufacturer. While the old version of the probe confirmed the previously reported low sensitivity (only 59% of the M. kansasii isolates reacted), the new one was 100% sensitive. Only two non-M. kansasii strains, both M. gastri isolates, gave false-positive hybridization results.

Isolation of an unusual mycobacterium from an AIDS patient.

A mycobacterium isolated from a clinical sample of an AIDS patient was identified as Mycobacterium interjectum by direct 16S rRNA sequence determination. High-performance liquid chromatography, however, revealed a mycolic acid pattern which was different from the one shared by the previously analyzed strains of this species.

Characterization of an isolate of the newly described species Mycobacterium interjectum.

The phenotypic features of a clinical isolate of the new species Mycobacterium interjectum, identified on the basis of its 16S rRNA gene sequence, are compared with those of the type strain. The differentiation of M. interjectum from Mycobacterium gordonae or Mycobacterium scrofulaceum is not achievable on the basis of phenotypic traits usually tested for mycobacterial speciation, but it can be reached by 16S rRNA gene sequencing or by high performance liquid chromatography (HPLC) of cell wall mycolic acids. The former reveals sequence identity with the signature region of the type species, and the latter yields a profile which is easily differentiated from those of the other two species. The unique HPLC profile of M. interjectum is reported here for the first time and so are the MICs of a wide spectrum of drugs.

Radiometric susceptibility testing of Mycobacterium xenopi.

Mycobacterium xenopi is an opportunistic pathogen, frequently isolated in various areas of Europe from pulmonary specimens, which may also cause infections in AIDS patients. We used the Bactec radiometric system (Becton Dickinson, USA) with a procedure expressly adapted to the particular growth characteristics of M. xenopi to determine the susceptibility patterns of 40 clinical isolates to six antimicrobial drugs. The majority of the strains were resistant to ethambutol and susceptible to amikacin, ciprofloxacin, rifampin, rifabutin and streptomycin.

Utility of high-performance liquid chromatography for identification of mycobacterial species rarely encountered in clinical laboratories.

High-performance liquid chromatography (HPLC) has been demonstrated to be a suitable technique for determining the species of mycobacteria on the basis of their mycolic acid pattern. Representative HPLC profiles, which are needed for the visual recognition of chromatograms, have been published for the most frequently encountered mycobacterial species. No extensive study has been reported for less common species, and only a few, scattered chromatographic patterns are available in literature. This study evaluates the utility of this technique for the identification of several rare species. Mycobacterium celatum, Mycobacterium genavense and Mycobacterium simiae chromatographic profiles have been verified, and previously unreported profiles of other species investigated. The chromatographic pattern of Mycobacterium malmoense is presented for the first time.

[Intestinal mycobacterial infections in AIDS. Clinical course and treatment of infections caused by Mycobacterium avium, Mycobacterium kansasii, Mycobacterium genavense]. / Micobatteriosi intestinali in AIDS. Clinica e terapia delle infezioni da Mycobacterium avium, Mycobacterium kansasii, Mycobacterium genavense.

Digestive apparatus is a common target of atypical mycobacteriosis in AIDS patients (at least 50% of patients with CD4+ lymphocytes < 50/mm3). We describe the clinical-histological features of two cases of Whipple-like syndrome likely caused by Mycobacterium avium (MAI) (study performed by light and electron microscopy), of one case of infection caused by two morphological variants of a MAI strain with a different sensitivity to antibiotics, of one case of M. kansasii infection and of two cases of M. genavense infection accompanied by sensitivity tests to antibiotics (as far as we know, these are the first described quantitative sensitivity tests of M. genavense to antibiotics). In conclusion, we discuss the present therapeutical outlines for M. kansasii and avium, together with the teramporary pharmacological options for M. genavense as suggested by antibiotic sensitivity tests performed on the strains isolated from the studied patients.

Investigation on several phenotypic features in two strains of Mycobacterium genavense.

The newly recognized species Mycobacterium genavense causes disseminated infections in AIDS patients, but its prevalence is difficult to assess because of its inability to grow on standard solid media. For the same reason, very little is known about the phenotypic traits of its isolates. We report here the results of our studies on two such strains isolated from AIDS patients and subcultured on a non-standard solid medium. Besides several features conventionally explored for mycobacterial speciation, we tested the isolates for 19 enzymatic activities and determined their mycolic acids profiles by means of high performance liquid chromatography. We also compare our findings with the scanty literature data on the laboratory characteristic and antimicrobial susceptibility of M. genavense.