Evaluation of spoligotyping-smear sputum as an alternative and culture-independent methodology for Mycobacterium tuberculosis genotyping

Abstract

Background: Tuberculosis (TB) remains as the second cause of death by an infectious disease preceded by the acquired immune deficiency syndrome (AIDS). Currently, laboratory techniques and methodologies of diagnosis and drug susceptibility testing are constantly changing. Therefore, it has been recommended the introduction of rapid assays based on the amplification of nucleic acids test (NAAT’s) through a polymerase chain reaction (PCR). Based on this principle, outstands spoligotyping - as a genotype and molecular epidemiology tool in tuberculosis – it is standardized to use isolated bacteria for the study of Mycobacterium genome through the amplification of 43 non-repetitive sequences, located at the direct repetitive region 1 (RD1).

Aim: Evaluation of spoligotyping from acid fast staining smears as an independent option from bacterial isolation to characterize Mycobacterium tuberculosis by using sputum samples from TB patients from National Cardiopulmonary Institute in Tegucigalpa, Honduras. Method: Of 37 patients with positive culture and smear microscopy for Mycobacterium, 50 expectoration samples were obtained. Microbiological and molecular tests were performed in respiratory samples containing mycobacterial DNA from sputum smears, concentrates and solid culture, for identification and genotype analysis by spoligotyping technique. Results: Spoligotyping was positive in 37/37 of positive culture samples (S: 100%), in 36/37 (S: 97.3%) of smear-positive samples and in 6/10 (S: 60%) of concentrate samples sputum. The intensity of positive smear microscopy had a direct relationship with the sensitivity of spoligotyping. Discussion: This study combined the potential of a molecular epidemiology tool to analyse DNA from sputum samples in smears acid fast staining, it visualizes diagnosis and genotyping platform in developing countries gathering innovation and high sensitivity in the hibridization of specific olignonucleotides from positive smears (P+, P++, P+++). However, the low specificity showed the need to improve better agreement among genetic patterns compared to the standardized bacterial isolation from M. tuberculosis strains.