in total , 165 blood and 194 sputum samples were collected from 181 different 
 hiv - positive patients with upper respiratory complaints , regardless of tb suspicions , 
 who were attended at the correia picano hospital and oswaldo cruz university hospital , 
 as shown in fig .
the samples were forwarded to 
 pernambuco central laboratory ( lacen - pe ) , where three unprocessed 0.5 ml - aliquots of 
 sputum were transferred into sterile microtubes , inside a biosafety cabinet located in 
 the mycology laboratory to assure no cross - contamination with cultivated 
 mycobacterium .
unprocessed sputum - aliquots and corresponding - patient 
 blood samples were then forwarded to the molecular biology laboratory at institute of 
 integrative medicine prof .
the remaining 
 sputum samples underwent smear microscopy and culturing at the reference laboratory for 
 tb diagnosis accredited by the brazilian ministry of health located in the lacen - pe .
all 
 samples were handled blindly by all technicians involved , from sample processing to 
 results analysis . 

microbiological analysis - acid - fast bacilli were detected by means of 
 ziehl - neelsen staining on sputa smears .
sputa were decontaminated in accordance with the 
 petroff method and aliquots of 0.1 ml were placed in lowenstein - jansen solid medium . 

genomic dna extraction - the patients blood samples underwent 
 ficoll - paque plus ( ge healthcare bio - sciences ab , sweden ) fractioning for leucocyte 
 isolation .
an aliquot of 10 leucocytes and the first of the three sputum 
 aliquots underwent genomic dna extraction using proteinase k protocol .
briefly , the 
 material was suspended in a 400 l lysozyme solution ( 1.25 mg / ml ) and was incubated at 
 37c for 16 h. then , 75 l of proteinase k ( 20 mg / ml ) and 10% sodium dodecyl sulfate 
 solution were added and the mixture was incubated at 65c .
next , 100 l of 5 m nacl and 
 100 l of ctab / nacl were added and the samples were incubated at 65c .
after 10 min of 
 incubation , the dna was extracted with phenol / chloroform solution and then concentrated 
 by means of precipitation with isopropanol .
the dna precipitate was obtained through 
 centrifugation at 16,000 g in a microcentrifuge , washed with 70% 
 ethanol and dissolved in 50 l of sterile water . 

conventional pcr - first , the genomic dna underwent pcr for 
 amplification of a target sequence on human dna , acting as a positive control for the 
 dna extraction , thereby ensuring the absence of pcr inhibitors in the reaction .
next , 
 pcr was performed with specific primers for amplification of the mycobacteria is6110 
 genetic element .
the total reaction volume was 50 l and included 1.5 mm of 
 mgcl2 , 25 pmol of each primer ( sense : 5cgagctgcgcgatggcgaac and 
 antisense : 5taggtgctggtggtccgaag , synthesised by invitrogen , usa ) , 200 m of each dntp 
 and 1.25 units of dna polymerase ( invitrogen ) .
the reaction conditions were one cycle of 
 96c for 3 min , 30 cycles of 96c for 30 s for denaturation , 65c for 30 s for 
 annealing , 72c for 30 s for extension and , finally , a cycle of 7 min at 72c using a 
 thermocycler ( gradient eppendorf , germany ) .
the reaction was considered positive when the 
 ethidium bromide - stained band corresponded to 245 bp . 

r 
 eal - time pcr - quantitative pcr ( qpcr ) was performed using the same set 
 of primers for is6110 amplification as used in conventional pcr and an internal probe 
 ( applied biosystems , usa ) in the presence of the taqman universal pcr master 
 mix and the dna extracted from the patients sputum or blood .
the conditions for 
 amplification of the is6110 element were as follows : denaturation for 15 min at 95c 
 followed by 45 cycles of 15 s at 94c and 60 s at 60c . in all pcr runs ,
standard curves 
 were obtained using plasmid dna encompassing the mycobacteria is6110 sequence as the 
 reaction positive control and all samples were tested in duplicate .
reaction with no dna 
 added was used as the negative control , also in duplicate .
the reaction was run in an 
 abi 7500 machine with setup for absolute quantification , using a standard curve prepared 
 with serial 10x dilution from 10 ng [ cycle threshold ( ct ) = 14 ] to 100 fg ( ct = 37 ) of 
 plasmid control dna in triplicate .
the efficiency of the amplification reaction was 1.8 , 
 with a threshold set at 0.02 based on data from 26 different experiments .
detect - tb - the second sputum aliquot was tested for the presence of 
 mycobacteria genome using the detect - tb ampligenix biotech company kit ( belo horizonte , 
 brazil ) .
briefly , the cells were lysed to release the genomic dna and to make it 
 soluble .
the extracted dna was purified by means of silica resin capture in the presence 
 of chaotropic agents , followed by successive washings and elution in a 
 low - ionic - strength solution .
pcr product - bounded to the is6110 
 immobilised probe was detected using a streptavidin - peroxidase / tetramethylbenzidine 
 colorimetric system .
readings within the absorbance range from 0.225 - 0.275 were 
 considered to be indeterminate .
values below 0.225 were considered to be negative and 
 above 0.275 as positive . 
 gen - probe amplified mtd - the third frozen stored sputum aliquot 
 underwent the gen - probe amplified mtd ( san diego , usa ) testing .
firstly , the 500 l 
 samples were defrosted at room temperature ( rt ) in a biological cabinet .
then , the same 
 volume of purifying solution of 2.9% sodium citrate and 4% naoh was added and incubated 
 for 15 min at rt .
the samples were neutralised by adding 500 l of phosphate buffer 
 solution ( ph 6.8 ) .
all reagents and equipment used in the gen - probe protocol were as recommended by the 
 manufacturer .
quality control - the flux and transport of the biological samples 
 between the different institutions involved in this project was carefully planned .
these 
 hospitals had a transport flow from their labs to the lacen - pe , which is responsible for 
 tb diagnosis in pe .
the main problem was identifying the samples related to this 
 research among the routine samples and therefore , we created small colourful forms to 
 send with the research samples , working with the personnel of the lacen - pe to identify 
 the samples related to this study , to avoid processing exams in duplicate and possible 
 specimen loss and to shorten the time of sample retention at the laboratory 
 reception .
another potential problem was related to where and how to aliquot the samples for the 
 different tests . from the outset
, we had decided to use the same samples for all tests 
 given the possibility of different samples having different bacterial loads even when 
 they are collected on the same sample day and the high likelihood that patients would 
 not return on the second day to bring the first - daily sputum . to avoid possible 
 cross - contamination with cultivated bacteria in the mycobacterium 
 facilities ,
the sputum samples were sent to the mycology lab because primarily 
 manipulation was performed inside a biological cabinet in that laboratory after working 
 hours .
three 0.5 ml - aliquots were prepared from each sputum specimen , which were frozen 
 and sent to the molecular biology laboratory at another institution under refrigeration ; 
 the remaining sputa were sent to the mycobacteria facilities at lacen - pe for 
 microbiological analysis .
all dna extractions were performed at the molecular biology laboratory of imip , inside a 
 biosafety cabinet located in a room with restricted use to clinic sample handling .
the 
 pcr reactions were prepared in another room specifically designed for this purpose , free 
 of biological samples and also inside a biosafety cabinet .
the hybridisation procedures 
 and the analysis of pcr products in gel were performed in the main area of the molecular 
 biology laboratory .
for every conventional or real - time pcr run , two negative controls were run : one 
 reaction was performed without the dna template and another included genomic dna of a 
 healthy individual to identify possible non - specific dna amplification . to avoid interpretation bias ,
the research teams at the hospitals , lacen - pe and the 
 molecular biology laboratory at imip , were different and the samples received a number 
 at the hospital , a different number at lacen - pe and yet another at the molecular biology 
 laboratory .
all patient identification numbers were noted in the patient database at the 
 molecular biology laboratory ; however , the codes were only broken for analysis at the 
 end of the cohort study . 

statistical analysis - the sensitivity , specificity and the positive 
 and negative predictive values ( npv ) for each of the molecular tests evaluated for the 
 diagnosis of tb in hiv - infected patients were calculated considering sputum culture as 
 the gold standard , using a 2 x 2 table .
the concordance between tests was evaluated 
 using the kappa ( k ) test ( epi info v.6.04 software ) .
the receiver operating 
 characteristic ( roc ) curves were performed using medcalc software , ostend , belgium . 

ethics - the aggeu magalhes research centre institutional review board 
 approved this study under the registration caae : 0007.0.095.000 - 07 .
the authors declare 
 no conflict of interest and do not have a direct financial relation with the commercial 
 identity mentioned in this manuscript .

 sputum samples - among 194 sputum samples from 181 different 
 hiv - infected patients , 19 were culture - positive for m. tuberculosis , of 
 which only eight were smear - positive . comparing the roc curves of the different tests with sputum culture as the reference , 
 the best results were associated with qpcr methodology ( fig .
qpcr presented 100% sensitivity , 96.7% specificity , 78.6% positive 
 predictive value ( ppv ) , 100% npv and 97% accuracy ( table i ) .
qpcr results demonstrated excellent concordance with culture ( k = 
 0.864 ) and moderate concordance with gen - probe results ( k = 0.586 ) ( table ii ) .
the ct average of culture - positive , 
 smear - positive sputa ( 32.5 ; range , 24.4 - 36.4 ) was similar to culture - positive , 
 smear - negative sputa ( 33.4 ; range 30.3 - 35.4 ) .
three samples presented culture - negative 
 and positive - qpcr results , with ct of 18 , 19 and 21 . 
 fig .
2 : receiver operating characteristic curve accuracy for molecular assay in 
 tuberculosis ( tb ) .
a : accuracy estimated for four different molecular assays 
 for tb testing in sputa using culture as gold - standard ; b : accuracy of 
 different cut - off set for tb testing in sputa using the detect - tb assay ; c : 
 accuracy of conventional e quantitative polymerase chain reaction ( qpcr ) for tb 
 diagnosis in blood . 
 

table ipositive ( ppv ) and negative ( npv ) predictive values , sensitivity and 
 specificity in samples using different molecular methods for the diagnosis of 
 tuberculosis ( tb ) and sputum culture as gold standardreference x tests ( sample)nppv ( % ) npv ( % ) sensitivity ( % ) specificity ( % ) culture x pcr ( sputum)19416.81 ( 9.4 - 29.5)100 ( 88.8 - 100)100 ( 67.9 - 100)46.3 ( 32.7 - 53.7)culture x qpcr ( sputum)10278.60 ( 48.8 - 94.3)100 ( 94.8 - 100)100 ( 67.9 - 100)96.70 ( 90 - 99.1)culture x detect - tb ( sputum)19424.36 ( 12.2 - 37)100 ( 91.6 - 100)100 ( 67.9 - 100)66.30 ( 47.4 - 68.3)culture x gen - probe ( sputum)10264.30 ( 35.6 - 86)97.70 ( 91.3 - 99.6)81.80 ( 47.8 - 96.8)94.50 ( 87.1 - 98)culture x pcr ( blood)16513.8 ( 8.3 - 21.7)95.9 ( 89.9 - 99.3)88.90 ( 63.9 - 98.3)32 ( 24.7 - 40.2)culture x qpcr ( blood)10261.1 ( 36.1 - 81.7)100 ( 94.6 - 100)100 ( 67.9 - 100)92.30 ( 84.3 - 96.6)detect - tb is a test in developing and gen - probe a commercial molecular test 
 for tb diagnosis .
pcr : in house polymerase chain reaction ; qpcr : quantitative real - time 
 pcr . 
 
detect - tb is a test in developing and gen - probe a commercial molecular test 
 for tb diagnosis .
pcr : in house polymerase chain reaction ; qpcr : quantitative real - time 
 pcr . 

table iiconcordance between microbiological and molecular tests for diagnosis of 
 tuberculosis ( tb)tests 
 ( sample)nagreementkappazpculture x pcr ( sputum)1940.4900.1392.760.0029culture x qpcr ( sputum)1020.9710.8648.800.0000culture x detect - tb ( sputum)1940.6280.2313.650.0002culture x gen - probe ( sputum)1020.9310.6826.950.0000culture x pcr ( blood)1650.3820.0621.830.0337culture x qpcr ( blood)1020.9310.7547.590.0000pcr ( sputum ) x qpcr ( sputum)1020.5200.1793.170.0007pcr ( sputum ) x detect - tb ( sputum)1020.6470.3013.150.0008pcr ( sputum ) x gen - probe ( sputum)1020.4800.1121.990.0236qpcr ( sputum ) x detect - tb ( sputum)1020.6570.2944.190.0000qpcr ( sputum ) x gen - probe ( sputum)1020.9020.5865.920.0000pcr ( blood ) x qpcr ( blood)1020.4610.1572.950.0016qpcr ( sputum ) x qpcr ( blood)1020.9020.6306.440.0000detect - tb is a test in developing and gen - probe a commercial molecular test 
 for tb diagnosis .
pcr : in house polymerase chain 
 reaction ; qpcr : quantitative real - time pcr . 
  detect - tb is a test in developing and gen - probe a commercial molecular test 
 for tb diagnosis .
pcr : in house polymerase chain 
 reaction ; qpcr : quantitative real - time pcr .
gen - probe presented the second - best results with 81.8% sensitivity , 94.5% specificity , 
 64.3% ppv , 97.7% npv and 93% accuracy .
a substantial concordance between gen - probe and 
 culture results was observed ( k = 0.682 ) detect - tb demonstrated a sensitivity of 100% , but a specificity of 66.3% , corresponding 
 to a ppv of 24.4% .
roc curves constructed with different cut - off values for testing 
 detect - tb showed that the best results for sensitivity and specificity occurred with a 
 cut - off of 300 ( 100% sensitivity , 67.4% specificity ) and 275 ( 100% sensitivity , 66.3% 
 specificity ) . in this study ,
the cut - off used was 275 , which was indicated by the 
 manufacturer ( fig .
however , higher cut - off 
 values might improve specificity with some compromised of sensitivity .
the lowest specificity was associated with conventional pcr performed from sputum 
 ( 46.3% ) and blood ( 32% ) samples ( table i ) . 

blood samples - concerning the molecular tests performed on the 
 patients blood samples , we only took into consideration samples from patients for whom 
 sputum also had been collected for definition of the tb cases based on sputum 
 culture . regarding blood samples
, qpcr showed 100% sensitivity , 92.3% specificity , 61.1% ppv and 
 100% npv in comparison to the sputum culture , whereas the in - house pcr showed 
 sensitivity and specificity values of 88.9% and 32% , respectively ( fig . 2c ) .
a substantial concordance was observed between qpcr 
 results obtained from blood and positive culture on sputum ( k = 0.754 ) and also between 
 qpcr results from blood and sputa ( k = 0.630 ) ( table 
 ii ) .
many studies on the accuracy of molecular assays have been published on the diagnosis of 
 pulmonary and non - pulmonary tb , both associated with smear - positive or smear - negative 
 sputum samples and in hiv - infected or non - infected patients ( peter et al .
smear - based microscopy is the most commonly used approach for pulmonary tb detection , 
 demonstrating 70% sensitivity compared with culture associated with a clinical 
 definition of the disease as the gold standard ; however , in hiv - infected patients the 
 smear - based sensitivity might be lower than 40% ( boehme 
 et al .
this was mainly related to the low bacillary load in sputum from 
 hiv - infected patients ( ogrady et al .
we observed a great variation in the accuracies of the four tests evaluated considering 
 that all were pcr - based assays and all used samples obtained from the same sputum .
qpcr 
 demonstrated superior performance with 97% accuracy , followed by gen - probe ( 93% ) , 
 detect - tb ( 70% ) and conventional pcr ( 52% ) for the detection of pulmonary tb infection 
 from sputum .
recent publications regarding the molecular detection of pulmonary tb in 
 hiv - infected patients also have shown variations in assay sensitivity ( 53 - 100% ) between 
 different or even the same assay ( table 
 iii ) . 

table iiiperformance of different molecular tests for tuberculosis detection in 
 hiv - infected patientsreferencehiv - infected 
 populationbiological 
 samplesampling ( n)methodologysensitivity ( % ) specificity ( % ) ppv ( % ) npv ( % ) target suspected 
 casesgold stantardthis paperrecife , brazilblood102qpcr10092.361.1100 in and outpatientsolid culturesputa102qpcr10096.778.6100102gene - probe81.894.564.397.7  detect - tb66.310024.4100albuquerque et al .
( 2013)malawi , africablood104xpert mtb / rif2110010064 in and outpatientsputum and solid cultureabed al - darraji et al .
( 2011)porto alegre , brazilsputa74pcr dot blot72858572 in and outpatientsolid culture combined with 
 clinicspcr ( agarose)42766853lawn et al .
( 2011)uganda , south africasputa and bronchoalveolar 
 lavage170mtb / rif3995 - -inpatientsolid culturehiv : human immunodeficiency virus ; npv : negative predictive value ; pcr : in 
 house polymerase chain reaction ; ppv : positive predictive value ; qpcr : 
 quantitative real - time pcr . 
 
hiv : human immunodeficiency virus ; npv : negative predictive value ; pcr : in 
 house polymerase chain reaction ; ppv : positive predictive value ; qpcr : 
 quantitative real - time pcr . for tb detection in sputum ,
our qpcr assay presented 100% sensitivity and 96.7% 
 specificity , comparable to the sensitivity of 80 - 95% and the specificity of 95 - 100% 
 reported by others who also developed the taqman assay for detection of the is6110 
 element ( gomez et al .
qpcr was positive for 100% of the smear - negative , culture - positive 
 patients , providing earlier tb diagnosis for more tb - suspected patients than smear - based 
 microscopy ( 7% vs. 4% ) in hiv - infected patients .
improvement in pulmonary tb detection 
 ( 25% ) by means of molecular assay was reported in chile , a tb non - endemic area , 
 confirming the power of molecular tools in tb diagnosis for tb - control programs ( balcells et al .
three patients with 
 false - positive qpcr from sputum received empirical treatment for tb within six months of 
 being tested and the clinical - symptom improvement confirmed their disease status .
the 
 withdrawal of a considerable ( 1.5 ml ) amount of sputum for molecular analysis prior to 
 sputum culturing might explained the absence of acid - fast bacillary growth on solid 
 media , likely due to the loss of bacillary viability as a consequence of the sputum 
 decontamination procedure ( michelon et al . 

2011 ) . the sensitivity and specificity of the gen - probe assay as reported in the literature 
 ranges from 92 - 100% and from 85 - 100% , respectively , but in hiv - positive patients the 
 sensitivity might be lower than 42% ( syre et al . 

, the performance of gen - probe was 
 similar to qpcr ( 93% vs. 97% accuracy ) , with 81.8% sensitivity and 94.5% specificity for 
 tb detection in sputum .
five false - positive cases were observed , two of whom received 
 empirical tb treatment with improvement in clinical symptoms .
in contrast to the qpcr 
 assay , the gen - probe assay was associated with two false - negative cases , possibly due to 
 the insufficient target dna in the 0.5 ml sputum aliquot used , considering that the 
 gene - probe protocol recommends the collection of 10 ml of sputum for decontamination and 
 concentration procedures prior to use .
the presence of pcr inhibitors in the sample may 
 also have induced false - negative reactions with the gen - probe assay , which were not 
 observed in the qpcr assay because , in this latter case , dna was extracted using 
 proteinase k digestion followed by a purification step performed with 
 phenol - chloroform .
cases identified as false - positives by the detect - tb assay were responsible for the low 
 specificity ( 66.3% ) and ppv ( 24.4% ) observed in our study .
the causes for this 
 performance are not clear , because a sensitivity of 75% and a specificity of 100% were 
 reported using the same assay for tb detection in spontaneous sputum samples obtained 
 from patients suspected of having tb ( michelon et al . 

difficulties in handling the kit , reagents stability and delays in 
 reading the reaction were considered .
the handling was performed exclusively by 
 laboratory personnel that had received training in executing the detect - tb protocol . to 
 read the samples
, we placed the microplates on a cold surface to transport them to 
 another laboratory in a connected building and thus some delay in reading might have 
 contributed to these results , although we also should consider that some of these 
 false - positive cases might indeed have been positive cases that had not been identified 
 by culturing .
a published meta - analysis demonstrated a sensitivity variation of 32 - 92% 
 with a mean of 72% and a specificity variation of 93 - 100% with a mean of 96% for the pcr 
 simplex methodology for tb detection , in comparison with sputum culture results and 
 clinical criteria ( sarmiento et al .
2003 ) . in our 
 study , we used only culture as the gold standard . nevertheless , the fact that qpcr and 
 conventional pcr results were so divergent surprises us because both assays used the 
 same primers and sample processing . the main differences were ( i ) qpcr used a 
 fluorescent internal probe for accuracy improvement , which per se 
 justifies this finding , while the conventional pcr did not have the probe hybridisation 
 step , ( ii ) samples were run in duplicate or triplicate in qpcr , whereas a single sample 
 normally was run for conventional pcr and ( iii ) qpcr data were measurable and positive 
 cases were defined based on a standard curve , whereas in conventional pcr , the results 
 were based on the identification of a band in agarose gel corresponding in size to the 
 amplified pcr product .
for all these reasons , the conventional pcr test may not be 
 reliable ( al zahrani et al . 2001 , chakravorty et al . 2005 ) .
the difference in sample processing is a limitation for comparative analysis of the 
 accuracy of different molecular assays for tb diagnosis .
a study on the performance of 
 the xpert mtb / rif test with an is6110-taqman qpcr assay for the detection of 
 mycobacteria in respiratory specimens processed by the standard procedure of 
 decontamination and centrifugation reported no differences in overall sensitivity ( 79% 
 vs. 84% , respectively ) of both tests , which presented 100% sensitivity for 
 smear - positive sputum , with no differences observed in smear - negative sputum sensitivity 
 ( 57% vs. 68% , respectively ) ( armand et al .
( 2013 ) compared the 
 performance of the xpert mtb / rif test using 1 ml of non - processed respiratory specimens 
 with the cobas taqman mtb assay using decontaminated - concentrated respiratory specimens 
 obtained from ~5 ml sputum .
the performance of both assays using culture results as the 
 gold standard showed that the overall sensitivities of the xpert ( 67.9% ) and cobas 
 ( 71.4% ) assays were similar .
the xpert sensitivity did not significantly differ between 
 smear - positive and smear - negative ( 67% vs. 69% ) sputum ; however , the reduction from the 
 87% sensitivity in smear - positive sputum to 54% sensitivity in smear - negative sputum 
 observed with the cobas assay suggested that the sputum - decontamination processing 
 included in the cobas protocol contributed to reducing the bacillary viability in 
 culture , even though the proportional sputum volume used was larger than that used in 
 the xpert test ( park et al . 2013 ) . comparing the performance of qpcr from sputum and blood samples of corresponding 
 patients , the qpcr test exhibited an accuracy of 93% for the detection of tb in blood , 
 showing good concordance with culture results ( 75% ) and the qpcr results from sputum 
 ( 63% ) .
one explanation for this higher concordance with sputum culture than with qpcr 
 testing of sputum was the high sensitivity of the qpcr test in sputum in relation to 
 culture .
the three cases of negative - culture and positive - qpcr results obtained from 
 sputum were also negative - qpcr for blood , suggesting that mycobacteraemia might be 
 associated with advanced disease . in this context ,
published data on the xpert test for 
 tb detection in blood revealed a 21% sensitivity and 100% specificity compared with 
 sputum culture ( 56% concordance ) , but a positive association was reported between 
 positive xpert assay from blood samples and the mortality rate in hiv - infected patients 
 ( feasey et al .
the main difference 
 between the xpert study and our protocol was in sample processing : we used leukocytes 
 purified from 5 ml peripheral blood , whereas the xpert protocol requires the collection 
 of 18 ml of peripheral blood for cellular lyses and concentration into a 1 ml sample for 
 introduction into the xpert cartridge for diagnosis . in this context , it is noteworthy 
 that blood components such as haem , lactoferrin and immunoglobulin g have been 
 associated with reduced amplification yield ( al - soud et 
 al .
2000 , al - soud & rdstrm 2001 ) 
 and the efficient removal of pcr - inhibitors from blood - extracted dna contributes to 
 improved sensitivity of the pcr - based diagnostic assay . in hiv - infected patients ,
confirmation of the diagnosis of pulmonary tb from sputum is 
 more difficult due to the low bacillary load in sputum and this difficulty is also 
 associated with external factors that influence the sample quality , such as inadequate 
 sputum collection , sample transportation , time delays in sample processing and the 
 methodology employed .
we believe that qpcr is one of the molecular tests that should be 
 considered as a tool for the detection of pulmonary tb in smear - negative patients .
 the present study analysed the concordance among four different molecular diagnostic 
 methods for tuberculosis ( tb ) in pulmonary and blood samples from immunocompromised 
 patients . 
 a total of 165 blood and 194 sputum samples were collected from 181 human 
 immunodeficiency virus ( hiv)-infected patients with upper respiratory complaints , 
 regardless of suspicious for tb . 
 the samples were submitted for smear microscopy , 
 culture and molecular tests : a laboratory - developed conventional polymerase chain 
 reaction ( pcr ) and real - time quantitative pcr ( qpcr ) and the gen - probe and detect - tb 
 ampligenix kits . 
 the samples were handled blindly by all the technicians involved , 
 from sample processing to results analysis . 
 for sputum , the sensitivity and 
 specificity were 100% and 96.7% for qpcr , 81.8% and 94.5% for gen - probe and 100% and 
 66.3% for detect - tb , respectively . 
 qpcr presented the best concordance with sputum 
 culture [ kappa ( k ) = 0.864 ) ] , followed by gen - probe ( k = 0.682 ) . for blood samples , 
 qpcr showed 100% sensitivity and 92.3% specificity , with a substantial correlation 
 with sputum culture ( k = 0.754 ) and with the qpcr results obtained from sputum of the 
 corresponding patient ( k = 0.630 ) . 
 conventional pcr demonstrated the worst results 
 for sputa and blood , with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% 
 vs. 32% , respectively . 
 commercial or laboratory - developed molecular assays can 
 overcome the difficulties in the diagnosis of tb in paucibacillary patients using 
 conventional methods available in most laboratories . 
SUBJECTS, MATERIAL AND METHODS
RESULTS
DISCUSSION