Laura Brum

pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates in Portugal

ABSTRACT The nucleotide sequences of the pncA genes within 55 multidrug-resistant pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates were determined. Fifty-three out of the 55 isolates were pyrazinamidase (PZase) negative. Four strains contained a wild-type pncA gene, and PZase activity was undetectable in two of these strains. Seven of the 18 identified pncA mutations found have not been described in previous studies.

Genetic characterisation of the ethambutol resistance-determining region in Mycobacterium tuberculosis: prevalence and significance of embB306 mutations

Ethambutol (EMB) is a first-line antitubercular drug that inhibits arabinogalactan and lipoarabinomannan biosynthesis. Resistance to EMB has traditionally been associated with embB mutations, especially in the Met306 codon. In this study, the region encompassing the embB306 codon in 109 Mycobacterium tuberculosis clinical isolates (49 EMB-susceptible and 60 EMB-resistant) was characterised. The occurrence of embB306 mutations was verified not only in EMB-resistant isolates (55.0%) but also in EMB-susceptible isolates (16.3%), which questions the role of embB306 mutations as determinants of EMB resistance. Subsequently, four different embB alleles were created by in vitro mutagenesis and were introduced into Mycobacterium smegmatis on a multicopy plasmid. To assess the contribution of embB306 mutations to EMB resistance, the EMB minimum inhibitory concentration (MIC) of these strains was determined. Strains carrying mutant embB306 alleles were able to grow at slightly higher MICs compared with the strain carrying the wild-type embB gene. The small MIC increase obtained here does not appear to be sufficient to cause high-level EMB resistance. The results obtained in the present study suggest that acquisition of EMB resistance might be a multistep process in which embB306 mutations may represent a first-step in EMB resistance acquisition.

Tuberculosis drug-resistance in Lisbon, Portugal: a 6-year overview.

Multidrug-resistance and extensive drug-resistance pose a serious threat to tuberculosis management in Portugal. The country has high TB incidence rates in comparison with other European Union countries, with the Lisbon Health Region being one of the most affected. In the present study we have analysed a convenience sample of 3025 Mycobacterium tuberculosis clinical isolates, recovered over a 6-year period (2001-2006) in the Lisbon Health Region, regarding drug-resistance both to first-line and second-line drugs. Moreover, 100 of these isolates were also genotyped by 12-loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeats (MIRU-VNTR) analysis. We have compared each year and observed the existence of 22 different resistance profiles, with MDR-TB rates ranging between 9.9% and 15.2% and XDR-TB rates, relative to the number of MDR-TB isolates, between 44.3% and 66.1% (excluding 1 year here considered as an outlier). A steady increase in the fraction of MDR-TB isolates resistant to all first-line drugs was also noticed. The genotyping analysis of MDR-TB isolates revealed six clusters, of which three (Lisboa3, Lisboa4 and Q1) were related to XDR-TB. Our results show that active transmission of MDR- and XDR-TB is taking place and that the high prevalence of observed XDR-TB is due to the continued transmission of particular genetic clusters. Enforcement of the implementation of genotyping in diagnostic routines would lead to early detection of resistant cases.

Genotypic analysis of Mycobacterium tuberculosis isolates from a Lisbon hospital in Portugal

Portugal has one of the highest tuberculosis notification rates of the European Union with Lisbon Health Region having an incidence rate well above the national average. The present study analyses the transmission, drug susceptibility and characteristics of a study population from a Central Lisbons Hospital. One hundred and thirty -two Mycobacterium tuberculosis clinical isolates were previously tested for drug susceptibility to first -line drugs. The multidrug (MDR) resistance rate was found to be 3.0%, while 13.6% of the isolates were resistant to one or more first -line drugs. HIV serology was available for 98 patients, 26 (26.5%) were positive. Genotyping was performed by MIRU -VNTR and 53 (40.2%) out of the 132 isolates were found to be distributed through 17 MIRU -VNTR clusters of two or more isolates. Lisboa strains accounted for 25.8% of all strains. We conclude that transmission of resistant and susceptible Mycobacterium tuberculosis strains is occurring, with special concern for Lisboa strains.

Vigilância laboratorial da resistência aos antibacilares em Portugal em 2000 - 2001

BACKGROUND: A network for the Surveillance of Antituberculosis Drug Resistance (VigLab-Tuberculose), including all the mycobacterial laboratories where drug susceptibility test on isolates of Mycobacterium tuberculosis complex are carried out, was established in Portugal in April 2000. VigLab-Tuberculose aims to maintain a laboratory-based surveillance system for antibiotic susceptibilities of Mycobacterium tuberculosis complex isolates in order to monitor trends in drug resistance in Portugal. OBJECTIVE: To describe the first line antituberculosis drug resistance patterns of tuberculosis cases diagnosed and reported to VigLab-Tuberculose in 2000-2001. METHODS: Collaborating laboratories collect and report data on individuals from whom a drug susceptibility test on Mycobacterium tuberculosis complex isolates has been performed in 2000-2001. Data collected included demographic, geographic, clinical and first line antibiotics susceptibility information. Data were analysed using Epi-Info version 6.04c software. RESULTS: There were 4170 reports of drug susceptibility test results on tuberculosis patients diagnosed from 1st April 2000 to 31st December 2001. Drug susceptibility results for all five first line antituberculosis drugs shows that 23% (541/ 2358) were resistant at least to one of them. The proportion of mono-resistance to streptomycin was 7,6% (179/2358), to isoniazid 2,6% (62/2358), to rifampicin 0,6% (15/2358) and to pyrazinamide 1,3% (30/2358). From the 4164 patients tested both to isoniazid and rifampicina, 244 (5,9%) were multidrug resistant. From patients with no history of previous tuberculosis treatment, 1,8% (28/1557) were mono-resistant to isoniazid, 0,4% (7/1557) to rifampicina, 4,2% (66/1557) to streptomycin and 1,7% (27/1557) to pyrazinamide. The proportion of primary multidrug resistance was 2,8% (43/1557) and acquired resistance was 13,3% (41/309). CONCLUSION: The laboratory participation rate was 80%, which is very encouraging for the first two years of VigLab-Tuberculose activities. The proportion of primary multidrug resistance was higher than the reported resistance from central and west Europe (less 1%), which reinforce the need and importance of maintaining and strengthening the laboratory-based surveillance in order to minimise the emergence of drug resistance. REV PORT PNEUMOL 2003; IX (4): 279-291

Molecular epidemiology of Mycobacterium tuberculosis in Lisbon

We conducted a molecular epidemiology study of Mycobacterium tuberculosis strains isolated from patients in Lisbon hospitals. We used restriction fragment length polymorphism (RFLP) to detect Lisbon family strains and to determine the genetic diversity of Mycobacterium tuberculosis strains isolated in Lisbon, through identification of the most important risk factors of tuberculosis transmission analysis, with the insertion sequence IS6110 as a probe to fingerprint isolates of Mycobacterium tuberculosis. 64.8% of the 290 Mycobacterium tuberculosis isolates were grouped in clusters. This figure was 60.7% if we excluded strains with five or fewer IS6110 copies. Multidrug-resistance was observed in 4.1% of the strains and they were all in clusters. Forty-five (18.2%) strains were included in the Lisbon family. Considering the relatively high percentage of strains in cluster detected in this study, we believe that active transmission is still taking place in Lisbon. Moreover, clusters of Lisbon strains represent the predominant strains circulating in Lisbon and are still related to drug resistance although presenting a lower percentage than that observed in previous studies.

Tuberculose multi-resistente. A propósito de um caso clínico de reinfeção exógena adquirida no meio prisional

RESUMO Os autores descrevem um caso clinic de tuberculose multi-resistente num doente recluse, toxicodependente e com SIDA que se encontrava sob terapeutiea antibacilar em virtude de anterior episodin de tuberculose “sendivel”. Comentam alguns aspectos relacionadus com o diagnostic, tratamento e evoucao exogena por estirpe multi-resistente. Em conclusao, us autores salientam o interesse do DNA “fingerprinting” no diagnostic e epidemiologia da tuberculose multi-resistente e a necessidade de implementacao de um efectivo programa de luta contra a tuberculose multi-resistente na comunidade prisional. REV PORT PNEUMOL 1998; IV (3): 319-325