Phialemoniopsis limonesiae sp. nov. causing cutaneous phaeohyphomycosis in an immunosuppressed woman.

Rare or opportunistic fungal infections are mostly described in immunosuppressed patients. We present a case of a cutaneous phaeohyphomycosis that developed on the dorsal foot in an immunosuppressed woman suffering from AIDS, caused by a novel Phialemoniopsis species. It clinically presented as an indurated violaceous plaque, surmounted by nodules exuding a sero-purulent discharge. A filamentous fungus was isolated from pus and cutaneous biopsy. ITS and LSU sequences phylogenetically resolved the fungus as an unknown species of Phialemoniopsis, which is an unresolved family within Sordariomycetes. In this study we describe the new species as Phialemoniopsis limonesiae, which clusters on a single branch clearly separated from its closest phylogenetic neighbours. This new strain showed low MIC to itraconazole, voriconazole and posaconazole.

[New tests for the diagnosis of tuberculosis]. / Nouveaux tests pour le diagnostic de la tuberculose.

Over the last decade, molecular biology techniques for identifying mycobacteria in pulmonary secretions have become more and more sensitive and rapid, even in smear negative samples. Nuclear amplification techniques also allow the rapid detection of resistance to first or second line anti-tuberculous drugs. The sensitivity of these techniques for non respiratory samples is yet to be determined. The diagnosis of latent tuberculous infection (LTBI) has also increased in sensitivity, specificity and positive predictive value through the use of interferon-γ release assays (IGRAs), which are tending to replace the tuberculin skin test, except for children aged under five. These tests, however, do have limitations which are important for the clinician; especially their inability to distinguish active from latent tuberculosis and their inability, in most circumstances, to exclude a diagnosis of active TB.

Posttraumatic ankle arthritis due to a novel Nocardia species.

INTRODUCTION: Nocardial arthritis in immunocompetent patients is rare, and the optimum duration of antimicrobial therapy is unknown, although several months of antibiotic treatment is often recommended. CASE REPORT: We here report the first case of human infection with a novel Nocardia sp., summarise the epidemiology of nocardial arthritis and outline the feasibility of relatively short antibiotic treatments after careful surgical drainage.

Tuberculosis cluster in an immigrant community: case identification issues and a transcultural perspective.

OBJECTIVES: In a low incidence area for tuberculosis (TB), a computerized database identified an unusually high proportion of patients coming from one single country between 2004 and 2006. To determine whether they constituted a cluster, whether clustering was due to recent transmission, and to understand what undermined the efficacy of the contact tracing procedure, we conducted a retrospective study of all patients with TB from this country. METHODS: Mycobacterium tuberculosis isolates of 15 TB cases originating from the same country over a 2(1/2) year period were analysed by restriction fragment length polymorphism (RFLP) and/or Rep-PCR. To identify links between patients, we revisited the social worker's files, cross-matched contacts' databases, and performed internet searches. A cultural evaluation was conducted by an anthropologist and an expert physician, through patient and community key informant interviews and a literature review. RESULTS: Genotyping confirmed that 11 of 15 patients had identical isolates. Additional data revealed an unsuspected complex network of social links between 9 of these 11 patients. The transcultural evaluation pointed out the major obstacles to efficient contact tracing, such as importance of social stigma related to TB, differences in communication style and health beliefs, and linguistic barriers. CONCLUSION: The combined finding of identical genotypes and important social links between patients confirmed the suspicion of a TB cluster due to recent transmission. The cultural evaluation helped to explain the difficulties encountered during the contact tracing procedure, and offered strategies to improve its efficacy despite the magnitude of the social stigma attached to TB in this community.

Assessment of a real-time PCR test to diagnose syphilis from diverse biological samples.

OBJECTIVES: To investigate the contribution of a real-time PCR assay for the detection of Treponema pallidum in various biological specimens with the secondary objective of comparing its value according to HIV status. METHODS: Prospective cohort of incident syphilis cases from three Swiss hospitals (Geneva and Bern University Hospitals, Outpatient Clinic for Dermatology of Triemli, Zurich) diagnosed between January 2006 and September 2008. A case-control study was nested into the cohort. Biological specimens (blood, lesion swab or urine) were taken at diagnosis (as clinical information) and analysed by real-time PCR using the T pallidum 47 kDa gene. RESULTS: 126 specimens were collected from 74 patients with primary (n = 26), secondary (n = 40) and latent (n = 8) syphilis. Among primary syphilis, sensitivity was 80% in lesion swabs, 28% in whole blood, 55% in serum and 29% in urine, whereas among secondary syphilis, it was 20%, 36%, 47% and 44%, respectively. Among secondary syphilis, plasma and cerebrospinal fluid were also tested and provided a sensitivity of 100% and 50%, respectively. The global sensitivity of T pallidum by PCR (irrespective of the compartment tested) was 65% during primary, 53% during secondary and null during latent syphilis. No difference regarding serology or PCR results was observed among HIV-infected patients. Specificity was 100%. CONCLUSIONS: Syphilis PCR provides better sensitivity in lesion swabs from primary syphilis and displays only moderate sensitivity in blood from primary and secondary syphilis. HIV status did not modify the internal validity of PCR for the diagnosis of primary or secondary syphilis.

Epidemiology of meningococcal disease in Switzerland, 1999-2002.

In Switzerland, immunisation against serogroup C meningococcal disease is recommended for persons at increased risk but is not included in the national vaccination programme. The aim of this study was to present the nationwide surveillance data on invasive meningococcal disease collected from 1999 to 2002, emphasising the evolution in the absence of extended vaccination. The number of reported cases of meningococcal disease peaked at 178 cases in 2000 (incidence rate of 2.5/100,000 person-years), with 61% of all cases attributed to serogroup C meningococci (incidence rate, 1.5/100,000 person-years). Since 2001, a spontaneous decrease in the reported cases was observed, resulting in an overall incidence rate of 1.4/100,000 person-years in 2002 (serogroup C cases, 0.8/100,000 person-years). On the other hand, the case-fatality rate of serogroup C cases increased to 18% in 2002, leading to an increase in the overall case-fatality rate from 8% to 14% (P>0.05). The small sample size reduces the interpretability of this observation. However, when the introduction of a generalised vaccination against serogroup C meningococcal disease is discussed, the fluctuations in the number of vaccine-preventable deaths should receive greater attention.

Severe Mycoplasma hominis infections in two renal transplant patients.

Systemic infections due to Mycoplasma hominis are rare and occur mainly in immunocompromised patients. Reported here are the cases of two renal transplant patients with peritonitis who did not respond to empirical antimicrobial treatment. Effective treatment with doxycycline was administered only after definitive identification of Mycoplasma hominis was achieved. For this identification, the new genetic amplification-sequencing method was invaluable.

Chlamydial serology: comparative diagnostic value of immunoblotting, microimmunofluorescence test, and immunoassays using different recombinant proteins as antigens.

To improve the reliability of the serodiagnosis of Chlamydia trachomatis infections, an immunoblot analysis, a microimmunofluorescence titration, and different immunoassays using synthetic peptides derived from species-specific epitopes in variable domain IV of the major outer membrane protein or recombinant antigens (heat shock protein 70 [hsp70], hsp60, hsp10, polypeptide encoded by open reading frame 3 of the plasmid [pgp3], macrophage infectivity potentiator, and a fragment of the total lipopolysaccharide) were evaluated. Because cross-reactions between chlamydial species have been reported, the microimmunofluorescence tests were also performed with Chlamydia pneumoniae and Chlamydia psittaci used as antigens, and C. pneumoniae-specific antibodies were also determined by immunoassays. Since the presence of antimicrobial antibodies must be interpreted in light of their prevalence in the general population, responses obtained with serum samples from patients with well-defined infection (i.e., with positive urethral or endocervical C. trachomatis DNA amplification) were compared to those obtained with samples from healthy blood donors. The best sensitivity (86%) with a specificity of 81% was obtained for immunoblotting results, when the number of individuals with > or =10 immunoglobulin G (IgG) and/or > or =2 IgM responses to the different C. trachomatis antigens was considered. A 13-kDa antigen was recognized by most of the samples (86% for IgG) from patients with acute urogenital infection but rarely (3%) by those from healthy blood donors (P < 0.0001). The sensitivity and specificity results obtained for serum antibodies to peptides or recombinant antigens were slightly lower than those results obtained for the number of responses to whole C. trachomatis antigens, which were 76 and 77%, respectively, when IgG responses to both recombinant hsp60 and pgp3 were considered.

Evaluation of the Bactec 960 automated nonradiometric system for isolation of mycobacteria from clinical specimens.

The Bactec MGIT 960 system (Becton Dickinson, USA), designed for the culture of mycobacteria, was compared with the Bactec 460 instrument and culture on two solid egg-based media using a total of 1024 clinical specimens. Mycobacteria could be identified from 99 (9.7%) specimens, 89 (90%) of which were identified by the Bactec 960 system, 90 (91%) by the Bactec 460 system, and 82 (83%) by culture on the two egg-based media. The Bactec 960 cultures became positive an average of 16.7 days after specimen collection, the Bactec 460 cultures 14.9 days after collection, and the cultures on egg-based media 26.2 days after collection. The Bactec 960 is a compact and highly automated nonradiometric system that may replace the Bactec 460 system.

Three cases of cutaneous sarcoidosis: search for bacterial agent by the 16S RNA gene analysis and treatment with antibiotics.

BACKGROUND: The aetiology of sarcoidosis remains controversial. An infectious origin is often discussed, but only anti-inflammatory or immunosuppressive treatment is recommended. OBJECTIVES: To investigate the hypothesis of bacterial origin by treating cutaneous sarcoidosis with antibiotics. METHODS: Patients with chronic cutaneous sarcoidosis, unresponsive to the usual treatment and not requiring systemic corticotherapy, were given combined antibiotherapy for 6 months. Search for bacterial DNA by amplification and sequencing of the 16S ribosomal RNA gene in skin biopsies of lesions before and after antibiotherapy was done. RESULTS: Three patients received a combined treatment with clarithromycin 1 g/day and ciprofloxacin 1 g/day. No clinical changes occurred in 2 cases and transient worsening in 1. Amplification for bacterial DNA was positive in all skin biopsies. The sequencing of this DNA could not identify a unique bacterial species. CONCLUSION: No evident bacterial origin could be demonstrated; however, this approach should be extended to more patients.

Detection of mycobacterial nucleic acids by polymerase chain reaction in fixed tissue specimens of patients with human immunodeficiency virus infection. Swiss HIV Cohort Study.

A polymerase chain reaction (PCR) method, which amplifies a fragment of the 16S ribosomal RNA (rRNA) gene present in all mycobacterial species, was developed and tested on 84 formalin-fixed paraffin-embedded tissue specimens from 51 patients with human immunodeficiency (HIV) infection. The PCR products were characterized either by sequencing or by hybridization with nonradioactive oligonucleotide probes specific for Mycobacterium tuberculosis complex, M. avium, or M. genavense. Sequencing was successful for 26 samples compared with the 45 samples for probe hybridization. The sensitivity of DNA amplification compared with microscopic examination was 79.5%. A mixed infection was detected with M. genavense for only one patient who was infected with M. tuberculosis complex. In the group of 22 control patients, where no diagnosis of mycobacterial infection was made during life and no acid-fast bacteria were seen during the autopsy, four samples of one patient were positive by hybridization with the M. tuberculosis probe. This patient had a clinical history compatible with tuberculosis. This PCR method may be a powerful tool for the precise diagnosis of mycobacterial infections from histopathologic material, provided that several sections from the same specimen block are tested.

Assessment of use of the COBAS AMPLICOR system with BACTEC 12B cultures for rapid detection of frequently identified mycobacteria.

The use of the COBAS AMPLICOR System (Roche Molecular Diagnostics, Basel, Switzerland), the only automated system for PCR testing, was evaluated for a rapid identification of mycobacteria with positive BACTEC 12B cultures. Two hundred ninety-six specimens with a growth index of >/=30 were analyzed for the presence of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare. Compared to traditional methods and provided that samples with PCR inhibition are retested at a 1:10 dilution, the sensitivity and specificity of the COBAS AMPLICOR System with BACTEC 12B cultures were 100 and 98%, respectively. The COBAS AMPLICOR method is rapid and reliable for identifying the most common mycobacteria in cultures.

Rapid diagnosis of pulmonary tuberculosis with the LCx Mycobacterium tuberculosis assay and comparison with conventional diagnostic techniques.

The LCx MTB amplification assay is a nucleic acid amplification test intended for the direct detection of Mycobacterium tuberculosis complex in respiratory specimens. We evaluated its performance on 2,001 consecutive respiratory specimens; 78 were culture positive for M. tuberculosis. Sensitivity, specificity, and positive and negative predictive values of this assay for all specimens compared to culture results were 88.5, 97.7, 60.5, and 99.5%, respectively. When referred to resolved clinical diagnosis of active tuberculosis, these values improved to 90.2, 98.4, 72.8, and 99.5%, respectively.

16S rRNA sequence diversity in Mycobacterium celatum strains caused by presence of two different copies of 16S rRNA gene.

Direct sequencing of the 16S rRNA gene (16S rDNA) of Mycobacterium celatum isolates showed ambiguities, suggesting heterogeneity. Cloned 16S rDNA yielded two copies of the gene, which differed by insertion of a thymine at position 214 and by additional mismatches. Restriction fragment length polymorphism analysis confirmed the presence of two copies of 16S rDNA within the bacterial chromosome.

Evaluation of the MB/BacT system and comparison to the BACTEC 460 system and solid media for isolation of mycobacteria from clinical specimens.

The MB/BacT automated system is designed for the isolation of mycobacteria from clinical specimens. It utilizes a colorimetric sensor and reflected light to continuously monitor the CO2 concentration in the culture medium. We compared its performance to that of the BACTEC 12B media for the radiometric BACTEC 460 instrument and that of solid culture media. Respiratory specimens and urine samples were decontaminated with 4% NaOH. The vials of the two instruments were inoculated with 500 microl of sample and two solid egg-based media at 200 microl each. All vials were incubated at 37 degrees C for 6 weeks. A total of 1,078 specimens (633 respiratory specimens, 78 cerebrospinal fluid specimens, 177 other body fluid specimens, 87 urine specimens, and 103 other types of specimens) were cultured in parallel. Mycobacteria could be identified from 73 (6.8%) specimens: 67 M. tuberculosis, 3 M. kansasii, 1 M. xenopi, 1 M. terrae, and 1 mixed M. avium with M. scrofulaceum. Of these, 63 (86.3%) specimens were positive with the MB/BacT system, 67 (91.8%) were positive with the BACTEC 460 instrument, and 58 (79.5%) were positive with the two egg-based media. MB/BacT cultures were positive on average after 17.5 (+/-6.4) days, BACTEC cultures with a growth index of >20 (mean, 200) were positive after 14.3 (+/-8.2) days, and egg-based media were positive after 24.2 (+/-7.5) days. Microorganisms other than mycobacteria contaminated 46 (4.3%) MB/BacT cultures and 31 (2.9%) BACTEC cultures, which had to be discarded. The MB/BacT system is a well-automated system for the detection of M. tuberculosis in clinical specimens without using radioactive reagents. Further trials are required to determine whether it is suitable for the culture of nontuberculous mycobacteria.

Evaluation of commercially available tests for Chlamydia nucleic acid detection in synovial fluid of patients.

Since the presence of Chlamydia nucleic acids has been shown in synovial fluid (SF) from some patients with Chlamydia reactive arthritis, we investigated whether commercially available tests, developed to detect Chlamydia nucleic acids in urogenital samples, could also be used for their detection in SF samples. We therefore tested SF samples, found positive with at least two different systems of DNA amplification in a previous study, with three commercially available kits. No positive results were obtained. It is concluded that the commercially available tests Gen-Probe PACE 2, Amplicor (developed by Roche Molecular Systems) and LCx (developed by Abbott Laboratories) do not have sufficient sensitivity to detect reliably Chlamydia RNA or DNA in SF.

Two different 16S rRNA genes in a mycobacterial strain.

Sequencing of the gene coding for 16S rRNA (16S rDNA) is a well-established method used to identify bacteria, particularly mycobacteria. Unique sequences allow identification of a particular genus and species. If more than one 16S rDNA is present on one mycobacterial genome, their sequences are assumed to be strictly or almost identical. We have isolated a slowly growing Mycobacterium strain, "X", identified by conventional biochemical tests as Mycobacterium terrae. Identification by amplification and direct sequencing of 16S rDNA yielded ambiguous results in two variable regions, suggesting the presence of different copies of the sequenced gene. Total DNA was digested by restriction enzymes and hybridized after Southern blotting to a probe representing about two-thirds of the 16S rDNA. Two copies of 16S rDNA were identified and cloned. By sequencing, the clones were of two different types, A and B, differing in 18 positions. Oligonucleotides specific to each copy of the 16S rDNA were used to distinguish the positions of the two genes observed in the Southern blot. We conclude that Mycobacterium strain "X" has two different copies of 16S rDNA. Variations in the sequence between two copies of 16S rDNA gene have been described in archaeobacteria, but not in mycobacteria. When placed in a phylogenetic tree together with other slowly growing mycobacteria gene A shows a common root with M. terrae, whereas gene B is placed separately.