Thioridazine Alters the Cell-Envelope Permeability of Mycobacterium tuberculosis.

The increasing occurrence of multidrug resistant tuberculosis exerts a major burden on treatment of this infectious disease. Thioridazine, previously used as a neuroleptic, is active against extensively drug resistant tuberculosis when added to other second- and third-line antibiotics. By quantitatively studying the proteome of thioridazine-treated Mycobacterium tuberculosis, we discovered the differential abundance of several proteins that are involved in the maintenance of the cell-envelope permeability barrier. By assessing the accumulation of fluorescent dyes in mycobacterial cells over time, we demonstrate that long-term drug exposure of M. tuberculosis indeed increased the cell-envelope permeability. The results of the current study demonstrate that thioridazine induced an increase in cell-envelope permeability and thereby the enhanced uptake of compounds. These results serve as a novel explanation to the previously reported synergistic effects between thioridazine and other antituberculosis drugs. This new insight in the working mechanism of this antituberculosis compound could open novel perspectives of future drug-administration regimens in combinational therapy.

Evaluation of the Capilia TB assay for culture confirmation of Mycobacterium tuberculosis infections in Zambia and South Africa.

The performance and cost of the Capilia TB assay were evaluated for use in a resource-limited setting. The sensitivity and specificity were 99.6% and 99.5%, respectively. The incremental costs of the Capilia test were estimated to be $1.46 and $1.84 when the test was added to liquid and solid culture processes, respectively. These findings suggest that the Capilia TB assay represents a rapid, simple, and inexpensive Mycobacterium tuberculosis identification test that can be used in resource-limited settings.

Mycobacterium noviomagense sp. nov.; clinical relevance evaluated in 17 patients.

Eighteen isolates of a nonchromogenic, slowly growing, non-tuberculous species of the genus Mycobacterium were cultured from respiratory specimens obtained over the last eight years from 17 patients in the Netherlands. These isolates were grouped because they revealed a unique 16S rRNA gene sequence and were related to Mycobacterium xenopi. None of the 17 patients met the American Thoracic Society diagnostic criteria for non-tuberculous mycobacterial disease, which distinguishes the novel isolates from the related species, M. xenopi. A polyphasic taxonomic approach, including identification by biochemical and phenotypical analysis, hsp65 gene sequencing and PCR restriction enzyme pattern analysis, and sequence analyses of the rpoB gene and 16S-23S internal transcribed spacer supported the separate species status of the novel isolates. The name Mycobacterium noviomagense sp. nov. is proposed for the novel strains. The type strain is NLA000500338(T) (=DSM 45145(T)=CIP 109766(T)). A more distinctive taxonomy of NTM is a prerequisite for the assessment of their clinical relevance.

Clinical relevance of Mycobacterium szulgai in The Netherlands.

BACKGROUND: The clinical relevance of Mycobacterium szulgai isolates is unknown, and available literature focuses on case reports of M. szulgai disease. We assessed the clinical relevance of M. szulgai isolated from patients in The Netherlands. METHODS: We reviewed medical files for all 21 patients in The Netherlands from whom M. szulgai was isolated during 1999-2006, applying the diagnostic criteria of the American Thoracic Society for nontuberculous mycobacterial infection. Random amplified polymorphic DNA genotyping was performed using IS986, OPA-2, and OPA-18 as primers. RESULTS: Of the 21 patients, 16 (76%) met the American Thoracic Society diagnostic criteria and were thus likely to have M. szulgai disease. Pulmonary M. szulgai disease was the most common presentation, with extrapulmonary disease restricted to patients with an impaired systemic immunity. Although treatment regimens varied in content and duration, the outcomes were mostly favorable. Both overtreatment and undertreatment were noticed. Random amplified polymorphic DNA genotyping revealed a higher degree of interpatient variability, with limited intrapatient variability, suggesting persisting monoclonal infection and good reproducibility. No genotype was associated with clinical relevance. CONCLUSIONS: Clinical isolation of M. szulgai generally represents true disease and demands careful follow-up. Extrapulmonary disease occurs in patients with impaired immunity. Adherence to diagnostic guidelines can be improved.

Tuberculosis outbreaks predicted by characteristics of first patients in a DNA fingerprint cluster.

RATIONALE: Some clusters of patients who have Mycobacterium tuberculosis isolates with identical DNA fingerprint patterns grow faster than others. It is unclear what predictors determine cluster growth. OBJECTIVES: To assess whether the development of a tuberculosis (TB) outbreak can be predicted by the characteristics of its first two patients. METHODS: Demographic and clinical data of all culture-confirmed patients with TB in the Netherlands from 1993 through 2004 were combined with DNA fingerprint data. Clusters were restricted to cluster episodes of 2 years to only detect newly arising clusters. Characteristics of the first two patients were compared between small (2-4 cases) and large (5 or more cases) cluster episodes. MEASUREMENTS AND MAIN RESULTS: Of 5,454 clustered cases, 1,756 (32%) were part of a cluster episode of 2 years. Of 622 cluster episodes, 54 (9%) were large and 568 (91%) were small episodes. Independent predictors for large cluster episodes were as follows: less than 3 months' time between the diagnosis of the first two patients, one or both patients were young (<35 yr), both patients lived in an urban area, and both patients came from sub-Saharan Africa. CONCLUSIONS: In the Netherlands, patients in new cluster episodes should be screened for these risk factors. When the risk pattern applies, targeted interventions (e.g., intensified contact investigation) should be considered to prevent further cluster expansion.

Molecular typing of Mycobacterium tuberculosis by mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis, a more accurate method for identifying epidemiological links between patients with tuberculosis.

IS6110 fingerprinting of Mycobacterium tuberculosis is the standard identification method in studies on transmission of tuberculosis. However, intensive epidemiological investigation may fail to confirm transmission links between patients clustered by IS6110-restriction fragment length polymorphism (RFLP) typing. We applied typing based on variable numbers of tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRUs) to isolates from 125 patients in 42 IS6110 clusters, for which thorough epidemiological data were available, to investigate the potential of this method in distinguishing epidemiologically linked from nonlinked patients. Of seven IS6110 clusters without epidemiological links, five were split by MIRU-VNTR typing, while nearly all IS6110 clusters with proven or likely links displayed conserved MIRU-VNTR types. These results provide molecular evidence that not all clusters determined on the basis of multibanded IS6110 RFLP patterns necessarily reflect transmission of tuberculosis. They support the use of MIRU-VNTR typing as a more reliable and faster method for transmission analysis.

Tuberculosis elimination in the Netherlands.

This study assessed progress towards tuberculosis (TB) elimination in the Netherlands by using DNA fingerprinting. Mycobacterium tuberculosis strains were defined as new if the IS6110 restriction fragment length polymorphism pattern had not been observed in any other patient during the previous 2 years. Other cases were defined as clustered and attributed to recent transmission. In the period 1995-2002, the incidence of TB with new strains was stable among non-Dutch residents and declined among the Dutch. However, the decline among the Dutch was restricted to those >or=65 years of age. Moreover, the average number of secondary cases per new strain did not change significantly over time. We conclude that the decline of TB in the Netherlands over the past decade was mainly the result of a cohort effect: older birth cohorts with high infection prevalence were replaced by those with lower infection prevalence. Under current epidemiologic conditions and control efforts, TB may not be eliminated.

Mycobacterium tuberculosis, Beijing genotype strains not associated with radiological presentation of pulmonary tuberculosis.

Mycobacterium tuberculosis strains of the Beijing genotype have been involved in various outbreaks of multidrug-resistant tuberculosis. Some studies suggest that the infection with the Beijing genotype is associated with a different host immune response. Since this might also lead to a different chest X-ray (CXR) presentation, we compared CXRs of patients with pulmonary tuberculosis, 33 of whom were infected with the Beijing genotype and 76 with other genotypes. No significant differences were detected. We conclude that the Beijing genotype is not associated with a different CXR presentation.

Molecular characteristics and global spread of Mycobacterium tuberculosis with a western cape F11 genotype.

In order to fully understand the global tuberculosis (TB) epidemic it is important to investigate the population structure and dissemination of the causative agent that drives the epidemic. Mycobacterium tuberculosis strain family 11 (F11) genotype isolates (found in 21.4% of all infected patients) are at least as successful as the Beijing genotype family isolates (16.5%) in contributing to the TB problem in some Western Cape communities of South Africa. This study describes key molecular characteristics that define the F11 genotype. A data-mining approach coupled with additional molecular analysis showed that members of F11 can easily and uniquely be identified by PCR-based techniques such as spoligotyping and dot blot screening for a specific rrs491 polymorphism. Isolates of F11 not only are a major contributor to the TB epidemic in South Africa but also are present in four different continents and at least 25 other countries in the world. Careful study of dominant compared to rare strains should provide clues to their success and possibly provide new ideas for combating TB.

Clustered tuberculosis cases: do they represent recent transmission and can they be detected earlier?

Clustered tuberculosis cases with Mycobacterium tuberculosis isolates showing identical restriction fragment length polymorphism patterns are assumed to be the result of disease transmission. In a prospective, population-based study in the province of North Holland, The Netherlands, we combined molecular methods with highly detailed epidemiologic information to determine why many clustered cases are not detected at an early stage. Of 481 patients, 138 (29%) fell into 43 clusters, suggesting recent transmission in 20%. Of 155 patients in clusters occurring within 2 years before or after the diagnosis of the disease, 21 (14%) had no epidemiologic links with other patients. Independent predictors of the absence of such links were female sex and Turkish, Moroccan, or other African ethnicity. Of 47 patients with a clear epidemiologic link, 37 (24% of 155) were identified early, e.g., by contact tracing, and 10 (6%) were missed. In 85 (55%) patients, an epidemiologic link was likely but undetected when using conventional contact tracing. Compared with clearly linked patients, only male sex was independently associated with presence in this last group. Our results indicate that 86% of clustered study patients had epidemiologic links and that opportunities for earlier identification using conventional tuberculosis control strategies are limited.

Nosocomial Mycobacterium bovis-bacille Calmette-Guérin infections due to contamination of chemotherapeutics: case finding and route of transmission.

We studied nosocomial infections due to Mycobacterium bovis bacille Calmette-Guérin (BCG) Onco-TICE bacteria, transmitted by contamination of medication prepared in BCG Onco-TICE-contaminated hoods in the pharmacy, in 5 immunocompromised patients at 3 hospitals. The BCG strains cultured from the patients had the same DNA profile as the BCG Onco-TICE strain used for bladder instillation. To prevent these infections, a change from open to closed preparation was made; strictly separated preparation in time of BCG Onco-TICE instillation and chemotherapy was enforced, the biological safety cabinet was disinfected between preparations, and gloves were changed between preparations.

False-positive mycobacterium tuberculosis cultures in 44 laboratories in The Netherlands (1993 to 2000): incidence, risk factors, and consequences.

False-positive Mycobacterium tuberculosis cultures are a benchmark for the quality of laboratory processes and patient care. We studied the incidence of false-positive cultures, risk factors, and consequences for patients during the period from 1993 to 2000 in 44 peripheral laboratories in The Netherlands. The national reference laboratory tested 8,889 M. tuberculosis isolates submitted by these laboratories. By definition, a culture was false positive (i) if the DNA fingerprint of the isolate was identical to that of an isolate from another patient processed within 7 days in the same laboratory, (ii) if the isolate was taken from a patient without clinical signs of tuberculosis, and/or (iii) if the false-positive test result was confirmed by the peripheral laboratory and/or the public health tuberculosis officer. We identified 213 false-positive cultures (2.4%). The overall incidence of false-positive cultures decreased over the years, from 3.9% in 1993 to 1.1% in 2000. Laboratories with false-positive cultures more often processed less than 3,000 samples per year (P < 0.05). Among 110 patients for whom a false-positive culture was identified from 1995 to 1999, we found that for 36% of the patients an official tuberculosis notification had been provided to the appropriate public health services, 31% of the patients were treated, 14% of the patients were hospitalized, and a contact investigation had been initiated for 16% of the patients. The application of DNA fingerprinting to identify false-positive M. tuberculosis cultures and the provision of feedback to peripheral laboratories are useful instruments to improve the quality of laboratory processes and the quality of medical care.

Mycobacterium microti infection (vole tuberculosis) in wild rodent populations.

Mycobacterium microti (vole tuberculosis) infections in small wild mammals were first described more than 60 years ago in several populations in Great Britain. Few studies of vole tuberculosis have been undertaken since then, and little is known about the relationship between M. microti isolates originating from different populations or at different times or of the prevalence of this infection in wild rodent populations, despite human cases of M. microti infections being increasingly reported. In this study, field voles (Microtus agrestis), bank voles (Clethrionomys glareolus), and wood mice (Apodemus sylvaticus) were found to be infected, with up to 8% having external tuberculous signs, in wild populations in Northumberland and Cheshire, England. Spoligotyping applied directly to the clinical material simultaneously detected and typed M. microti bacteria in skin lesions, lymph glands, and internal abcesses. IS6110 restriction fragment length polymorphism typing of cultured bacteria was used to compare these isolates with previously isolated strains from both animals and humans. This demonstrated that although the current rodent isolates were distinct from those isolated from voles in the 1930s in Great Britain, they had a high degree of similarity to these strains and were distinct from the M. microti isolates from humans, a pig, and a ferret from The Netherlands. Thus, M. microti infection seems to be widespread in wild rodent populations, but more studies are needed to understand how M. microti might be transmitted from animals to humans and to determine better the zoonotic risk posed.