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Mycobacterium tuberculosis strains are becoming resistant to not only the most powerful first-line drugs but also many second-line drugs., Mycobacterium tuberculosis strains that are resistant to an increasing number of second-line drugs used to treat multidrug-resistant tuberculosis (MDR-TB) are becoming a threat to public health worldwide. We surveyed the Network of Supranational Reference Laboratories for M. tuberculosis isolates that were resistant to second-line anti-TB drugs during 2000–2004. We defined extensively drug-resistant TB (XDR-TB) as MDR-TB with further resistance to ≥3 of the 6 classes of second-line drugs. Of 23 eligible laboratories, 14 (61%) contributed data on 17,690 isolates, which reflected drug susceptibility results from 48 countries. Of 3,520 (19.9%) MDR-TB isolates, 347 (9.9%) met criteria for XDR-TB. Further investigation of population-based trends and expanded efforts to prevent drug resistance and effectively treat patients with MDR-TB are crucial for protection of public health and control of TB.

Buruli ulcer, caused by Mycobacterium ulcerans, is a chronic ulcerative skin disease, found predominantly in central and west Africa and Australia. A boy of 2.5 years of age from Angola was admitted to our hospital with severe kwashiokor and a large ulcer with undermined edges on the left side of the thorax. Further examination revealed anaemia, hypoproteinaemia, bacterial superinfection of the ulcer and intestinal parasites. Histological analysis showed acid-fast bacilli and histopathological changes typical of Buruli ulcer. M. ulcerans was detected by PCR and culture. The patient was treated by surgical excision of diseased skin, followed by split-skin grafting. He also received antibiotic therapy (ciprofloxacin, clarithromycin, rifabutin, and dapsone). After six months, the child was discharged from hospital in good condition. This is the first published case of Buruli ulcer from Angola.

Although Europe identified the pathway to tuberculosis (TB) elimination in 1990, no information on programmes for country preparedness is available. A questionnaire investigating TB elimination activities was submitted to 38 national TB programme representatives of low TB incidence (

The ability of fluorescence resonance energy transfer, molecular-beacon, and TaqMan probes to detect single nucleotide polymorphism (SNP) in the presence of a wild-type allele was evaluated using drug resistance-conferring SNPs in mixed Mycobacterium tuberculosis DNA. It was found that both the absolute quantity and the ratio of alleles determine the detection sensitivity of the probe systems.

Ethambutol (EMB) is in use worldwide as a first-line anti-tuberculosis drug and substitutions in codon 306 of the embB gene are the most common mutations found in EMB resistant Mycobacterium tuberculosis (MTB) strains. Pyrosequencing is a real time sequencing method able to rapidly detect mutations in a large number of samples. Using this technique we analyzed, in parallel with conventional sequencing, a 24 bp region of the embB gene of 28 MTB clinical isolates. Pyrosequencing efficiently identified all embB306 mutations, detecting three different single-base substitutions leading to 2 amino acid changes (Met to Val or Ile). Mutated embB alleles were detected in 2 multidrug-resistant (MDR) EMB-susceptible strains. Overall, our results demonstrated that the Pyrosequencing method efficiently recognizes mutations in embB in a very short time and represents a valid molecular method to detect mutations in the MTB embB306 region.

[for full text, please go to the a.m. URL], GMS Zeitschrift zur Förderung der Qualitätssicherung in medizinischen Laboratorien; 4:Doc01; ISSN 1869-4241

Attempts to improve the diagnosis of tuberculosis (TB) in high-burden countries has resulted in significant funding and initiatives to change the method of diagnosis of TB from light microscopy supplemented with X-ray to a sophisticated diagnostic algorithm based on the latest technological innovations. Such activities are overdue and should be welcomed, but the lack of skills and support available to interpret and use the results represents a danger. The introduction of new diagnostic methods, particularly liquid culture, should be carefully structured according to the local situation, failing which frustration and the disruption of previously underdeveloped but adequately functioning laboratories may result

Nine Kazakhstan oblasts, 2001.To analyse the genetic relationship of drug-resistant Mycobacterium tuberculosis strains from Kazakhstan and to determine the frequency of the Beijing genotype.All drug-resistant smear-positive cases identified in nine oblasts during the 2001 nationwide drug resistance survey were analysed by IS6110 fingerprinting and spoligotyping. Isolates were obtained from 150 patients (64 new and 86 retreated cases).Eight cases (5.3%) of dual infection were identified. Of the remaining 142 strains, 91 (64.1%) were grouped in 18 clusters, indicating a high rate of recent transmission of resistant tuberculosis (TB). This was further confirmed by the origin of the patients as well as by the similar drug resistance patterns of the clustered strains. Accordingly, more than one third of all clustered strains were new cases. About 70% of the resistant strains belonged to the Beijing genotype, compared to only 37.5% in a control group of 40 susceptible isolates.Transmission of drug-resistant strains seems to contribute to the spread of resistant TB in this high incidence region. The Beijing genotype should be seen as a major cause of drug-resistant TB in Kazakhstan and was found to be associated with drug resistance.

Germany, 1995 to 2001.To determine the genetic relationship of 451 multidrug-resistant (MDR) Mycobacterium tuberculosis strains from Germany and to identify strains of the Beijing genotype.All strains were analysed using IS6110 fingerprinting and a cluster analysis was performed. Clustering of isolates was used as a measure for recent transmission.Two hundred and fourteen of 433 strains (49.4%) with more than four IS6110 copies formed 46 fingerprint clusters comprising two to 32 patients. Transmission links based on classical epidemiological data could be established for 39 cases (18.2%) and in 14 clusters (30.4%), and included three cases of exogenous reinfection with MDR strains. One hundred and seventy-five strains (38.8%) were of the Beijing genotype with an increasing annual proportion from 19.2% in 1995 to 58.3% in 2001. About 70% of these patients had an indication of foreign birth, mainly the former Soviet Union.Transmission of MDR strains seems to be contributing to the spread of MDR-TB in Germany, and exogenous reinfection with MDR strains must be considered as a possible cause of treatment failure. A high proportion of these MDR strains is probably carried over from the former Soviet Union, and strains of the Beijing genotype represent an increasing cause of MDR-TB in Germany.

Data regarding possible differences in microbiological response to therapy of disease caused by Mycobacterium tuberculosis and M. africanum are limited. Presenting clinical characteristics and sputum bacillary load during standard short-course chemotherapy in patients with newly-diagnosed pulmonary tuberculosis due to M. tuberculosis (n = 7) and M. africanum (n = 6) were compared. Changes in sputum bacillary load were measured using quantitative acid-fast bacilli smears, colony forming unit assay, and time until positive culture in the BACTEC radiometric system. Presentation and response to short course chemotherapy were comparable between patients infected with M. tuberculosis and those infected with M. africanum.

The molecular prediction of isoniazid (INH) resistance in Mycobacterium tuberculosis is hampered by the need for specialized equipment, expertise, high costs, a limited range of detectable mutations, or several of these factors. The rationale for the study was to find a practical alternative and to demonstrate generally valid problems.DNA extracted from decontaminated sputum pellets was used to amplify a 0.26 kb target sequence within the katG gene. Mutations of codon 315, frequently found in isoniazid-resistant isolates, could be discriminated in a simple agarose minigel format following an AciI digest of the nested polymerase chain reaction (PCR) product. Within a panel of 22 sputum samples, INH resistance could be predicted in 5 of 10 samples containing isoniazid-resistant M. tuberculosis. The protocol is robust, requires little expertise and no specialized equipment, and provides the test results within 2 days.The results show the feasibility to rapidly and easily detect mutations highly predictive of isoniazid resistance. Nevertheless, this, like any other molecular resistance prediction test, is affected by often neglected factors, including mutation prevalences, the phenomenon of heteroresistance, and a possible bias toward one's own method.

Initial isolates should be tested for drug susceptibility to confirm the anticipated effectiveness of chemotherapy.To evaluate E-test strips for susceptibility testing of Mycobacterium tuberculosis.A proportion method using Lowenstein-Jensen medium and the Bactec radiometric system were compared with the E-test (isoniazid [INH], rifampicin [RMP], ethambutol [EMB] and streptomycin [SM]).For 73 of the 81 M. tuberculosis isolates (90.1%) the proportion and E-test methods yielded concordant susceptibility results against all four antimicrobial agents tested. Of these 73 strains, 69 were fully susceptible; the four isolates showing resistance to antimicrobial drugs by both methods were also resistant when tested by Bactec 460TB. While the proportion method indicated susceptibility for the eight remaining strains, E-test results showed mono EMB resistance in five strains, INH resistance for two isolates (including one isolate resistant to EMB plus INH), and for one strain E-test yielded resistance to EMB and SM. Using Bactec as the reference method, the E-test resulted in false resistance in eight strains and no false susceptibility.Due to a substantial rate of false resistance, this method cannot be recommended at present for practical use in clinical laboratories.