Genotypes of drug-resistance Mycobacterium tuberculosis and its relationship with their drug resistance level and the mutations in the North Region of Buenos Aires Province

IMPERIALE BELÉN; ZUMÁRRAGA MARTÍN ; DI GIULIO BEATRÍZ; CATALDI ANGEL; MORCILLO NORA

Bucaramanga

Congreso; International Congress. Tuberculosis, Leprosy and Mycobacteriosis: ?Back to the biblical times: today solutions?. VI meeting of the SLAMTB 2012; 2012

Universidad Industrial de Santander, Colombia

Imperiale B1, Zumárraga M2, Di Giulio B3, Cataldi A2, Morcillo N1. 1. Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr. Cetrángolo Hospital, Vicente Lopez, Buenos Aires Province. 2. Tuberculosis Laboratory, Biotechnology Institute, National Institute of Agricultural Technology, Castelar, Buenos Aires Province. 3. Tuberculosis Laboratory, Petrona V de Cordero Hospital, San Fernando, Buenos Aires Province. belen.imperiale@conicet.gov.ar; mzumarraga@cnia.inta.gov.ar; betydiyu@fibertel.com.ar; acataldi@cnia.inta.gov.ar; nora_morcillo@yahoo.com.ar Introduction: Nowadays, almost 500.000 new cases of multidrug-resistant tuberculosis (MDR-TB) occur each year and about 5-7% of them will evolve to extensively drug-resistant TB (XDR-TB). Considering this, the aim of the study was to characterize drug resistance (DR) clinical isolates according to their genetic patterns and its relationship with isoniazid (INH), rifampicin (RIF) and levofloxacin (LX) resistance levels and the specific mutations conferring resistance. Materials and methods: Clinical specimens from period 2006-2012 were decontaminated and concentrated by the mix NaOH and N-Acetyl-L-Cistein or the NaOH-NaCl (hypertonic solution) and cultivated into solid media and MGIT960. Drug susceptibility testing to INH, RIF and LX was performed by the indirect proportion method on Löwenstein Jensen and the MGIT 960 system. DR level was determined by the minimal inhibitory concentration (MIC) using the micro plate colorimetric method. A multiplex allele specific PCR and DNA sequencing were used to detect mutations conferring DR. Genotyping of the isolates was performed by spoligotyping and IS6110 RFLP. Results: In this study were incorporated 219 M. tuberculosis clinical isolates: 78 mono-resistant to INH (INH-R; 1 also resistant to LX); 13 mono-resistant to RIF (RIF-R); 7 to LX, 71 MDR (3 also LX-R); 50 fully drug-susceptible (DS) and the Reference strain H37Rv ATCC 29274. Of the INH-R isolates, 54.0% had mutations in katG315, 25.5% in inhAP-15, while 20.5% didn?t show mutations in this fragments. Within INH-R isolates containing mutations in katG315, 94.7% showed intermediate to high DR level (MIC_INH >32.0-2.00 µg/mL), and 5.3% a low level resistance (1.00-0.50 µg/mL). Half part of the isolates with mutation in inhAP-15 presented low MIC values. Regarding to RIF-R isolates, 62.0% had mutations in rpoB531, 17.0% in rpoB526 and 5.0% in rpoB516. Besides, 73.5% of the isolates with mutation in rpoB531 showed high DR level (MIC_RIF ≥64.00 µg/mL), 16.3% intermediate levels (MIC_RIF: 8.0-4.0 µg/mL), and only 10.2% low levels resistance (MIC_RIF ≤2.0 µg/mL). Fifty percent of the mutated isolates in rpoB526 had intermediate to high RIF-R level. Three LX-R isolates presented gyrA94 mutation with high MIC values (16.00-4.00 µg/mL) and other one had a mutation in gyrA90 with intermediate LX-R levels (MIC_LX: 0.50 µg/mL). Spoligotyping patterns belonging to Haarlem (H), LAM and T were the most relevant families found in the study. H and LAM were the most related to katG315, and T to inhAP-15. Isolates mutated in rpoB531 were homogeneously distributed within the three spoligotyping families, and those mutated in rpoB526 were distributed between H and T. Conclusions: KatG315 mutation: related to intermediate-high DR levels, MDR and H and LAM; inhAP-15 mutation: related to low INH-R levels and mono-resistance to INH and T family. RpoB531-516 mutation: related to high DR level, H, LAM and T.