tuberculosis ( tb ) is caused by the mycobacterium tuberculosis , a member of the m. tuberculosis complex ( mtbc ) . according to the world health organization ( who ) , in 2010 , there were an estimated 12.0 million prevalent cases of tb ( equivalent to 178 cases per 100000 individuals ) and approximately 1.4 million people died of tb during that year . most of the cases in 2009 ( 55% ) occurred in asia ( 1 ) . according to the  administration of tuberculosis and leprosy control  of the ministry of health and medical education in iran , in 2010 , a total of 10485 old and new cases of tb were reported in iran and of these cases , 326 patients ( around 2.2% ) were hiv positive ( 2 ) . the diseases caused by mycobacterium complex have become more important in the recent years , particularly in association with the worldwide pandemic situation caused by human immunodeficiency virus ( hiv ) . in 2010 , in addition , there were an estimated 0.35 million deaths among tb cases that were hiv - positive . according to who , the incidence of pulmonary tuberculosis in hiv - positive patients in iran was 0.38 ( 0.28 - 0.51 ) per 100000 individuals in 2012 ( 3 ) . some opportunistic microorganisms such as nocardia spp . are gram - positive ( weakly acid fast ) and filamentous with a branching appearance and are not part of the normal human bacterial flora . to date at least sixteen species capable of producing disease in humans have been identified ( 4 ) . pulmonary disease is the predominant clinical presentation of this microorganism and can be fatal if untreated . untreated pulmonary nocardiosis is similar to tuberculosis and n. asteroids is the most frequent cause of pulmonary infection in humans ( 85% ) ( 5 ) . since the clinical and radiological manifestations are non - specific , pulmonary nocardiosis could be mistaken with other infections or other bacterial pneumonia . in addition , nocardiosis may be under reported because it is not among the aids - defining criteria ( 6 ) . diagnosis of pulmonary nocardiosis is dependent on the isolation or demonstration of the organism from respiratory secretions such as sputum or tissue specimens . nocardia as a m. tuberculosis is a slow growing organism that requires prolonged incubation for at least 4 - 6 weeks ( 7 ) . the diagnosis of nocardiosis is currently based on direct examination and conventional culture ; serology is usually not useful . treatment of nocardiosis is different from tuberculosis regarding therapeutic agents , route of administration and duration of therapy ( 8) . sulfonamides have been the agents of choice for the treatment of nocardiosis , however combination therapy with two or more agents is recommended . although nocardiosis is an uncommon illness , co - infection of this disease with tuberculosis has been reported ( 9 ) . the aim of this study was to assess the presence of nocardia spp . and m. tuberculosis in sputum specimens of patients with pulmonary tuberculosis . in the meantime , 32 hiv - infected patients who were suspected of having pulmonary tuberculosis were included in this study in order to evaluate concomitant infection in these patients . a total of 189 sputum samples were obtained from the reference mycobacteriology laboratory , ahvaz jundishapur university of medical sciences from march 2011 to april 2012 . all specimens were processed immediately according to standard routine diagnosis procedures by the naoh / n - acetyl - l - cysteine ( nalc ) method . next , concentrated specimens were used for smear preparation , culture and pcr assay ( 10 , 11 ) . furthermore , 250 l of each concentrated sputum specimen was inoculated onto lowenstein - jensen ( l - j ) media ( merck , germany ) . are acid fast and can survive the decontamination of clinical specimens with sodium hydroxide and nalc methods , thereby , they can grow on l - j medium . cultures were examined weekly and after 8 weeks of incubation , cultures , which showed no growth , were reported negative and discarded . for microscopic examination , using a disposable pasteur pipette , one drop of the sediment of the sputum specimen was spread on each clean microscope slide and after air drying and fixation by heat , the smears were stained by ziehl - neelsen and gram staining ( 10 ) . after decontamination , the chromosomal dna was extracted using proteinase k and phenol : chloroform , and then precipitated by ethanol and isopropanol , according to standard procedures ( 12 ) . the purified dna was amplified with specific pairs of primers is1 ( 5ctcgtccagcgccgcttcgg 3 ) and is2 ( 5cctgcgagcgtaggcggtgg 3 ) for m. tuberculosis complex strains ann microbiol . the pcr protocols were optimized to 35 cycles consisting of 45 seconds at 94c for denaturation , 30 seconds at 68c for annealing and 30 seconds at 72c for extension , followed by a final 5 minute extension at 72c . primers ng1 ( 5ctcgtccagcgccgcttcgg3 ) and ng2 ( 5cctgcgagcgtaggcggtgg3 ) were used to amplify a nocardia genus specific 590-bp fragment of 16s rrna . the pcr protocols were optimized to 40 cycles consisting of 45 seconds at 94c for denaturation , 30 seconds at 57c for annealing and 30 seconds at 72c for extension , followed by a final 5 minute extension at 72c . amplification with these primers was observed by electrophoresis on 2% ( w / v ) agarose gel stained with ethidium bromide ( figure 1 ) . the n. asteroides atcc 19247 and m. tuberculosis h37rv were used as positive reference strains . two pcrs for each sample were performed in separate tubes with two pairs of primers . one set was dedicated to the m. tuberculosis complex , whereas the other set was for nocardia spp . for each round of pcr the investigators were blinded to clinical data during the experiment for detection of microbial agents co - infection . all specimens were unnamed and labeled by a secure code in the reference mycobacteriology laboratory . a : clinical sample of m. tuberculosis . a total of 189 sputum samples were obtained from the reference mycobacteriology laboratory , ahvaz jundishapur university of medical sciences from march 2011 to april 2012 . all specimens were processed immediately according to standard routine diagnosis procedures by the naoh / n - acetyl - l - cysteine ( nalc ) method . next , concentrated specimens were used for smear preparation , culture and pcr assay ( 10 , 11 ) . furthermore , 250 l of each concentrated sputum specimen was inoculated onto lowenstein - jensen ( l - j ) media ( merck , germany ) . are acid fast and can survive the decontamination of clinical specimens with sodium hydroxide and nalc methods , thereby , they can grow on l - j medium . cultures were examined weekly and after 8 weeks of incubation , cultures , which showed no growth , were reported negative and discarded . for microscopic examination , using a disposable pasteur pipette , one drop of the sediment of the sputum specimen was spread on each clean microscope slide and after air drying and fixation by heat , the smears were stained by ziehl - neelsen and gram staining ( 10 ) . after decontamination , the chromosomal dna was extracted using proteinase k and phenol : chloroform , and then precipitated by ethanol and isopropanol , according to standard procedures ( 12 ) . the purified dna was amplified with specific pairs of primers is1 ( 5ctcgtccagcgccgcttcgg 3 ) and is2 ( 5cctgcgagcgtaggcggtgg 3 ) for m. tuberculosis complex strains ann microbiol . the pcr protocols were optimized to 35 cycles consisting of 45 seconds at 94c for denaturation , 30 seconds at 68c for annealing and 30 seconds at 72c for extension , followed by a final 5 minute extension at 72c . primers ng1 ( 5ctcgtccagcgccgcttcgg3 ) and ng2 ( 5cctgcgagcgtaggcggtgg3 ) were used to amplify a nocardia genus specific 590-bp fragment of 16s rrna . the pcr protocols were optimized to 40 cycles consisting of 45 seconds at 94c for denaturation , 30 seconds at 57c for annealing and 30 seconds at 72c for extension , followed by a final 5 minute extension at 72c . amplification with these primers was observed by electrophoresis on 2% ( w / v ) agarose gel stained with ethidium bromide ( figure 1 ) . the n. asteroides atcc 19247 and m. tuberculosis h37rv were used as positive reference strains . two pcrs for each sample were performed in separate tubes with two pairs of primers . one set was dedicated to the m. tuberculosis complex , whereas the other set was for nocardia spp . for each round of pcr the investigators were blinded to clinical data during the experiment for detection of microbial agents co - infection . all specimens were unnamed and labeled by a secure code in the reference mycobacteriology laboratory . a : clinical sample of m. tuberculosis . among the 157 outpatients , 7.6% ( 12/157 ) of specimens had positive results for acid fast bacilli by acid fast staining but none of the samples were positive for nocardia spp . after cultures evolution , 10.1% ( 16/157 ) of specimens had positive results for mtbc while no specimens grew nocardia spp . during microscopic examination by the gram staining method , we could nt find any particle morphologically similar to nocardia . out of 157 samples , 7% ( 11/157 ) were positive by pcr for mtbc and none of the samples was positive for nocardia by pcr . among 32 samples in hiv - infected patients , four ( 12.5% ) were culture positive for m. tuberculosis and none of them were positive for nocardia . furthermore , these four samples were positive by acid fast staining and pcr assay . out of these four samples , two were positive for nocardia spp . by the pcr method . based on our data the prevalence of nocardiosis and also concomitant infection with tuberculosis in our study was 6.25% ( 2/32 ) in hiv - infected patients and 1.05% ( 2/189 ) for all cases . among the 157 outpatients , 7.6% ( 12/157 ) of specimens had positive results for acid fast bacilli by acid fast staining but none of the samples were positive for nocardia spp . after cultures evolution , 10.1% ( 16/157 ) of specimens had positive results for mtbc while no specimens grew nocardia spp . during microscopic examination by the gram staining method , we could nt find any particle morphologically similar to nocardia . out of 157 samples , 7% ( 11/157 ) were positive by pcr for mtbc and none of the samples was positive for nocardia by pcr . among 32 samples in hiv - infected patients , four ( 12.5% ) were culture positive for m. tuberculosis and none of them were positive for nocardia . furthermore , these four samples were positive by acid fast staining and pcr assay . out of these four samples , two were positive for nocardia spp . by the pcr method . based on our data the prevalence of nocardiosis and also concomitant infection with tuberculosis in our study was 6.25% ( 2/32 ) in hiv - infected patients and 1.05% ( 2/189 ) for all cases . as clinical manifestation of pulmonary tuberculosis and nocardiosis are similar , laboratory tests are required in order to distinguish of nocardiosis from tuberculosis . nocardia is a slow growing bacteria and the contamination risk of the culture with other bacteria and fungi is high . therefore , the suggested approach for this problem is molecular techniques , since they are more sensitive than conventional diagnostic methods ( 15 ) . as two samples were positive for nocardia spp . and mtbc using the pcr method , it is suggested that concurrent pulmonary tuberculosis and nocardiosis may developed coincidently . nocardiosis has been reported to be more prevalent in hiv infected patients . in these patients , infection with opportunistic agents such as nocardia is attributed to suppression of cell mediated immunity ( 16 ) . it has been suggested that , the most common condition predisposing patients to nocardiosis is underlying chronic lung disease . in this study it was found that the coincidence of pulmonary tuberculosis and nocardiosis was 1% for the entire study population and 6.25% among hiv - infected patients . the incidence of pulmonary nocardiosis in hiv - infected patients has been previously reported by some investigators . pulmonary nocardiosis in hiv - infected patients with suspected pulmonary tuberculosis was reported to be 3% by alnaum et al . ; they reported an average of 3 - 4% for this infection ( 17 ) . some reports indicate that greater than two - thirds of the patients diagnosed with pulmonary nocardiosis were initially diagnosed as having tuberculosis and about 5% of the patients with proven pulmonary tuberculosis were shown to have co - infection with nocardia ( 18 ) . out of 140 sputum samples from african hiv - positive individuals clinically suspected of having tuberculosis ( tb ) , the frequency of nocardiosis was reported as 3.6% ( 19 ) . in areas where hiv - associated tb is common , only a few case reports of concomitant infection of nocardia and tb have been published in the literature ( 20 ) . a prevalence of 1.4% for pulmonary nocardiosis was reported in a tuberculosis and chest diseases hospital in amritsar by singh et al . although , pintado et al . indicated that the overall incidence of nocardiosis among hiv - infected patients is between 0.1 and 0.4% , and is associated with high morbidity and mortality rates ( 18 ) . concurrent pulmonary aspergillosis and nocardiosis was reported in an immunocompromised patient following long - term steroid therapy ( 22 ) . in this study nocardia was not distinguished in sputum specimens using conventional methods , however , positive samples were determined using the pcr assay . our study showed that the pcr technique was more sensitive than conventional methods in detection of nocardia . accordance of molecular techniques with conventional methods has been reported as 70 to 90% by several studies ( 22 , 23 ) .   in iran , several studies investigated pulmonary nocardiosis or tuberculosis in patients with various clinical symptoms . recently , aminzade et al . reported concomitant pulmonary nocardiosis and tuberculosis in a patient with rheumatoid arthritis ( 24 ) . in a study by eshraghi and amin , nocardia asteroides was isolated from only one patient suffering from cushing s syndrome with bronchogenic carcinoma amongst 142 patients with advanced symptomatic pulmonary disease in tehran , iran ( 25 ) . in another study from iran ( shojaei et al . ) clinical isolation of n. cyricigeorgica from patients with various clinical manifestations was performed ( 26 ) . to the best of our knowledge , this is the first study reporting co - infection of nocardiosis and tuberculosis in hiv - positive patients in iran . concurrent pulmonary tuberculosis and nocardiosis is much more frequent in hiv - infected patients and can be fatal . although nocardiosis resembles tuberculosis , first line anti - tuberculous drugs are not efficient for its treatment . therefore , it is important to establish a potent diagnosis method with high sensitivity and specificity such as molecular methods to improve the speed of diagnosis of nocardiosis .