Françoise Portaels

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology

BackgroundThe Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.ResultsThe fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network.ConclusionOur results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Resazurin Microtiter Assay Plate: Simple and Inexpensive Method for Detection of Drug Resistance in Mycobacterium tuberculosis

ABSTRACT A method for detecting multidrug-resistant Mycobacterium tuberculosis by using a reduction of resazurin is described. Eighty clinical isolates were evaluated against isoniazid and rifampin; results at 7 days were compared with those of the proportion method. Specificity and sensitivity were excellent. The method is simple, inexpensive, and rapid and might be used with other antituberculosis drugs.

Worldwide emergence of extensively drug-resistant tuberculosis.

Mycobacterium tuberculosis strains are becoming resistant to not only the most powerful first-line drugs but also many second-line drugs.

Epidemiology of antituberculosis drug resistance (the Global Project on Anti-tuberculosis Drug Resistance Surveillance): an updated analysis.

BACKGROUND The burden of tuberculosis is compounded by drug-resistant forms of the disease. This study aimed to analyse data on antituberculosis drug resistance gathered by the WHO and International Union Against Tuberculosis and Lung Disease Global Project on Anti-tuberculosis Drug Resistance Surveillance. METHODS Data on drug susceptibility testing for four antituberculosis drugs--isoniazid, rifampicin, ethambutol, and streptomycin--were gathered in the third round of the Global Project (1999-2002) from surveys or ongoing surveillance in 79 countries or geographical settings. These data were combined with those from the first two rounds of the project and analyses were done. Countries that participated followed a standardised set of guidelines to ensure comparability both between and within countries. FINDINGS The median prevalence of resistance to any of the four antituberculosis drugs in new cases of tuberculosis identified in 76 countries or geographical settings was 10.2% (range 0.0-57.1). The median prevalence of multidrug resistance in new cases was 1.0% (range 0.0-14.2). Kazakhstan, Tomsk Oblast (Russia), Karakalpakstan (Uzbekistan), Estonia, Israel, the Chinese provinces Liaoning and Henan, Lithuania, and Latvia reported prevalence of multidrug resistance above 6.5%. Trend analysis showed a significant increase in the prevalence of multidrug resistance in new cases in Tomsk Oblast (p<0.0001). Hong Kong (p=0.01) and the USA (p=0.0002) reported significant decreasing trends in multidrug resistance in new cases of tuberculosis. INTERPRETATION Multidrug resistance represents a serious challenge for tuberculosis control in countries of the former Soviet Union and in some provinces of China. Gaps in coverage of the Global Project are substantial, and baseline information is urgently required from several countries with high tuberculosis burden to develop appropriate control interventions.

Pulmonary Tuberculosis in HIV-Infected Patients in Zaire — A Controlled Trial of Treatment for Either 6 or 12 Months

BACKGROUND We studied the efficacy of a short-course regimen of chemotherapy for pulmonary tuberculosis in Kinshasa, Zaire. We also assessed whether, among patients with human immunodeficiency virus (HIV) infection, treatment should be extended from 6 to 12 months. METHODS HIV-seropositive and HIV-seronegative outpatients with pulmonary tuberculosis were treated with rifampin, isoniazid, pyrazinamide, and ethambutol daily for two months, followed by rifampin plus isoniazid twice weekly for four months. The HIV-positive patients who had no evidence of tuberculosis were then randomly assigned to receive either rifampin plus isoniazid or placebo twice weekly for a further six months. We also followed a comparison group of HIV-seronegative patients who received no further treatment for tuberculosis after six months. RESULTS After six months, 260 of 335 HIV-seropositive and 186 of 188 HIV-seronegative participants could be evaluated, and their rates of treatment failure were similar: 3.8 and 2.7 percent, respectively. At 24 months, the HIV-seropositive patients who received extended treatment had a relapse rate of 1.9 percent, as compared with 9 percent among the HIV-seropositive patients who received placebo for the second 6 months (P < 0.01). Extended treatment did not improve survival, however. Among the HIV-seronegative patients, 5.3 percent relapsed. CONCLUSIONS Among HIV-seropositive patients with pulmonary tuberculosis, extending treatment from 6 to 12 months reduces the rate of relapse but does not improve survival. The six-month program of partly intermittent antituberculous treatment may be an acceptable alternative when resources are limited.

Insects in the transmission of Mycobacterium ulcerans infection

Buruli ulcer, caused by Mycobacterium ulcerans, is a common disease of skin and bones, which usually occurs in the vicinity of rural tropical wetlands. Epidemiological studies have not established a natural reservoir or mode of transmission. We collected 30 plants from swamps in areas of Benin (Ouinhi and Zangnanado) and Ghana (Tontokrom) endemic for Buruli ulcer. We tested roots, stems, and leaves for M ulcerans by culture and nested PCR. , 3 We also studied five water bugs found on the roots of three of these plants. Of the 95 samples analysed, only the five insects were positive by PCR for M ulcerans. These insects were from the roots of plants belonging to the C y p e r u s, P a n i c u m, and E i c h h o r n i a genera. Two of them from the roots of the C y p e r u s a n d P a n i c u m were not identified but were probably water bugs of the family Naucoridae. Roots of E i c h h o r n i a sheltered three aquatic bugs, one belonging to the genus N a u c o r i s ( f a m i l y Naucoridae) and two to the genus D i p l o n y c h u s ( f a m i l y Belostomatidae). Only the insects were positive; not the roots, stems, or leaves. Three different genera of plants are involved, suggesting that the type of plant is not a factor, but it is the aquatic bugs in the roots that are of interest. These bugs are aggressive predators of other species of aquatic arthropods and molluscs. They fly from one nearby swamp or pond to another, and may bite human beings. Water bugs belonging to the generae N a u c o r i s and Diplonychus often bite villagers. A study of ten water bugs from the same areas showed that two were positive for acid-fast bacilli. Only occasional acid-fast bacilli were seen, suggesting that water bugs were mechanical vectors of M ulcerans. We propose a new hypothesis for a source of M ulcerans a n d one mode of its transmission to human beings. M ulcerans survives best under low oxygen tensions, such as exist in mud in the bottom of swamps. Some water-filtering organisms could concentrate M ulcerans and be ingested by waterdwelling predators, which are then a passive reservoir for M ulcerans. Bites of these insects or contamination of human skin by their faeces may explain why direct contact with water is not necessary to acquire Buruli ulcer. Trauma seems essential for the introduction of M ulcerans into the skin. T h e aetiological agent may be introduced directly by bites of these insects, or by trauma at skin sites contaminated by insect products containing M ulcerans. There may also be parallel modes of transmission of M ulcerans, such as by aerosols from the surface of swamps. If confirmed, we believe this would be the first implication of insects in the transmission of a mycobacterial disease.

Rapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay

SETTING Multidrug resistant Mycobacterium tuberculosis strains are threatening TB control in the world. Rapid diagnosis of resistance is essential for adequate treatment and optimal control of the disease. OBJECTIVE Evaluation of a new technique (Line Probe Assay, LiPA) for easy and rapid detection of Rifampicin resistance (RMPR) of M. tuberculosis. DESIGN After amplification of the region of the RNA polymerase, involved in RMPR, the amplified product is hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip. From the pattern obtained the presence or absence of RMPR M. tuberculosis can be assessed. 67 clinical samples positive in culture for M. tuberculosis were analyzed with LiPA and results were compared with classical susceptibility testing. RESULTS In vitro drug sensitivity testing identified 46 rifampicin sensitive and 21 resistant strains. In 65 of the 67 specimens LiPA results matched classical testing. In two RMPR cases LiPA showed a sensitive pattern. CONCLUSION In contrast to culture and sensitivity testing, where results take on average 6 weeks, LiPA testing is an easy and rapid (< 48 h) method of detecting RMPR M. tuberculosis in clinical samples. Results correlated in 97% of the samples. In the two RMPR samples with a sensitive LiPA pattern another mechanism of resistance is suspected.

Resazurin Microtiter Assay Plate Testing of Mycobacterium tuberculosis Susceptibilities to Second-Line Drugs: Rapid, Simple, and Inexpensive Method

ABSTRACT The emergence of multidrug-resistant tuberculosis calls for new, rapid drug susceptibility tests. We have tested 150 Mycobacterium tuberculosis isolates against the second-line drugs ethionamide, kanamycin, capreomycin, ofloxacin, and para-aminosalicylic acid by the colorimetric resazurin microtiter assay and the proportion method. By visual reading, MICs were obtained after 8 days. A very good correlation between results by the colorimetric resazurin microtiter assay and the proportion method was obtained. The colorimetric resazurin microtiter assay is inexpensive, rapid, and simple to perform, and implementation of the assay is feasible for low-resource countries.

Buruli Ulcer (M. ulcerans Infection): New Insights, New Hope for Disease Control

Buruli ulcer is a disease of skin and soft tissue caused by Mycobacterium ulcerans. It can leave affected people scarred and disabled. What are the prospects for disease control?

Mycobacterium ulcerans disease

Mycobacterium ulcerans disease (Buruli ulcer) is an important health problem in several west African countries. It is prevalent in scattered foci around the world, predominantly in riverine areas with a humid, hot climate. We review the epidemiology, bacteriology, transmission, immunology, pathology, diagnosis and treatment of infections. M. ulcerans is an ubiquitous micro-organism and is harboured by fish, snails, and water insects. The mode of transmission is unknown. Lesions are most common on exposed parts of the body, particularly on the limbs. Spontaneous healing may occur. Many patients in endemic areas present late with advanced, severe lesions. BCG vaccination yields a limited, relatively short-lived, immune protection. Recommended treatment consists of surgical debridement, followed by skin grafting if necessary. Many patients have functional limitations after healing. Better understanding of disease transmission and pathogenesis is needed for improved control and prevention of Buruli ulcer.