Detection and characterization of nocardia from patients diagnosed as tuberculosis in egypt

Abstract

Pulmonary tuberculosis and pulmonary nocardiosis are similar in most clinical symptoms and radiological manifestation. In the developing countries like Egypt where tuberculosis is very common, anti-tuberculosis drugs are started on basis of radiology and clinical symptoms. This study included 600 sputum specimens collected from 200 patients diagnosed as pulmonary tuberculosis from three chest hospitals in Egypt. IS6110 specific primer were selected for PCR to identify the Mycobacterium tuberculosis and hsp65 gene specific primers were used for PCR and sequencing for the identification of Mycobacterium and Nocardial species. The region of the gene coding for 16S rRNA in Nocardia species were selected as genus specific primer sequences for a PCR and Real Time PCR assays. Our result confirmed that four whole DNA samples, extracted from sputum specimen from the pulmonary tuberculosis patients on anti-tuberculosis treatment, were Nocardia species. Three of them matched (99% homology) with Nocardia farcinica (formerly Nocardia asteroid type V) and one match (83% homology) with Nocardia pneumonia. Molecular methods such as PCR and real-time PCR for identification of Nocardia are rapid and accurate methods. No cross-reactions were observed using Real Time PCR specific for Nocordia with other closely related genera.

Keywords: 16S rRNA; Mycobacterium tuberculosis; Nocardia; real time PCR.

Figure 1

Agarose gel electrophoresis of positive 540 bp PCR products of IS6110 gene. Lane M, 1 kbp DNA ladder; Lane 1, M. tuberculosis H37Rv; Lanes 2-10, Random DNA samples from the 200 clinical sputum samples.

Figure 2

Agarose gel electrophoresis of positive 440bp PCR products of hsp65 gene. Lane M, 1 kbp DNA ladder; Lane 1, M. tuberculosis; Lanes 2-5, Nocardia species; Lanes 6-13, Mycobacterium species.

Figure 3

Agarose gel electrophoresis of PCR products of 16S rRNA gene by using primers NG1 and NG2 specific for detection of Nocardia species. Lane M, 1 kbp DNA ladder; Lane 1, M. tuberculosis H37Rv; Lanes 2-5, positive 16S rRNA 595 bp of Nocardia species from clinical samples; Lane 6, DNA from M. tuberculosis clinical isolates; Lane 7, DNA extracted from Mycobacterium chelonae.

Figure 4

Detection of Nocardia species by real time PCR SYBR Green. Delta Rn, Display dye fluorescence as a function of cycle number; 1, negative (H₂O); 2-5, positive with NG1 and NG2 primer specific for genus Nocardia; 6, M. tuberculosis H37Rv; 7, DNA from M. tuberculosis.

Figure 5

Melting curve analysis by real time PCR. Derivatives, displays a plot of the first derivative of the rate of change in fluorescence as a function of temperature; 1, negative (H₂O); 2-5, positive with NG1 and NG2 primer specific for genus Nocardia; 6, M. tuberculosis H37Rv; 7, DNA from M. tuberculosis.