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 .
 background : tuberculosis ( tb ) remains as one of the most serious infectious diseases in the world . 
 pulmonary tuberculosis can occur with other pulmonary diseases caused by opportunistic organisms such as nocardia spp . particularly in immunocompromised patients . 
 therefore , diagnosis of co - infection at the early stage of the disease could be lifesaving.objectives:the goal of this study was to detect mycobacterium tuberculosis and nocardia spp . in sputum specimens in order to assess the concomitant nocardiosis and tuberculosis in patients with suspected pulmonary tuberculosis.patients and methods : from march 2011 to april 2012 , 189 sputum specimens were obtained from patients who were suspected of having pulmonary tuberculosis . 
 out of 189 samples , 32 of the samples belonged to hospitalized hiv - infected patients . 
 samples were examined by gram and ziehl - nelsen staining , culture and pcr methods.results:from 157 sputum specimens , positive samples by acid fast staining , culture and pcr for m. tuberculosis were reported for 7.6% ( 12/157 ) , 10.1% ( 16/157 ) and 7% ( 11/157 ) of samples , respectively . 
 no results were obtained by the described methods for nocardia spp . among 32 samples of hiv - infected patients , four ( 12.5% ) had positive results for acid fast staining , culture and pcr detecting m. tuberculosis while only two samples had positive results for nocardia spp . by pcr and no results 
 were reported by culture , gram and acid fast staining for this organism.conclusions:concurrent pulmonary nocardiosis and tuberculosis is frequent in hiv - infected patients . 
 rapid and sensitive methods such as pcr are recommended for detection of such co - infections . 
1. Background
2. Objectives
3. Patients and Methods
3.1. Sputum Collection and Processing
3.2. DNA Extraction and PCR Assay
4. Results
4.1. Specimens
5. Discussion