Patent Application: US-56622303-A

Abstract:
the present invention relates to a method of processing clinical samples for diagnosing bacterial infections by smear microscopy , culture or pcr as well as a kit for performing the said method . this invention also pertains to two sets of primers specific for the devr gene of m . tuberculosis as well as to a method of using said primers for screening patients for tuberculosis by pcr .

Description:
a multipurpose method has been developed for laboratory diagnosis of tuberculosis by smear microscopy , culture and pcr . the method is eminently suitable for the preparation of smears for highly sensitive microscopy . it effectively decontaminates samples making them suitable for culture and yields good quality inhibitor - free mycobacterial dna for use in pcr - based diagnostic assays . it also enables the isolation of good quality mycobacterial rna from clinical specimens employing common rna isolation procedures . the method for processing of sputum samples for smear microscopy , culture and pcr is as follows : ( i ) solution 1 : universal sample processing solution ( usp ): 4 - 6 m guanidinium hydrochloride ( guhcl ), 50 mm tris - cl , ph 7 . 5 , 25 mm edta , 0 . 5 % solution of n - lauroylsarcosine ( henceforth referred to as sarcosyl ), 0 . 1 - 0 . 2 m β - mercaptoethanol . ( 4m guhcl and 0 . 1 m β - mercaptoethanol is preferred when only culture / culture and smear are to be performed ; 5m guhcl and 0 . 1 - 0 . 2 m β - mercaptoethanol is preferred in cases where culture is performed together with smear microscopy and pcr ; 6m guhcl and 0 . 2 m β - mercaptoethanol is preferred in cases where only smear microscopy , pcr and rt - pcr are done ). ( ii ) solution 2 : 68 mm sodium phosphate buffer , ph 6 . 8 ( optional ). ( iii ) sterile water ( preferably double or triple distilled ); ( depc treated - sterile water in case of rna isolation . ( iv ) solution 3 : resuspension solution ( 0 . 05 % tween 80 in sterile water ). for isolation of dna from low bacillary load samples , the following additional reagents are needed : ( i ) solution a : 10 % suspension of a resin , which is sodium salt of paired iminodiacetate ions coupled to styrene divinylbenzene matrix ( henceforth referred to as chelex 100 ). ( ii ) solution b : 0 . 03 % triton x - 100 . ( iii ) solution c : 6 . 3 % tween 20 . note : for the isolation of dna from smear positive samples and sterile fluids , solutions a , b and c are not required ; amplifiable dna may be isolated by directly heating the sample in solution 3 at 90 - 100 ° c . for 30 - 40 minutes ( or solution 3 may be supplemented with triton x - 100 at a final concentration of 0 . 01 % ( see fig1 ). 1 ) to the sputum sample add 1 . 5 to 2 volumes of solution 1 in a 50 ml screw capped vial . 2 ) if possible , resuspend the sputum in usp solution using disposable plastic pasteur pipette of 5 ml capacity . 3 ) vortex well avoiding frothing ( as much as possible ) till the sputum is well homogenised ( this is generally accomplished within 30 - 40 seconds ). complete homogenisation is necessary for optimal results ( for highly purulent large volume and tenacious and hemoptyic sputum containing blood clots , incubation for 10 minutes at room temperature after vortexing may necessary ). 4 ) keep standing for 5 - 10 minutes at room temperature and add 10 - 15 ml of solution 2 to the homogenate . this step aids in formation of a proper pellet . mix gently and spin at 4000 - 5000 rpm at room temperature for 10 - 15 minutes in a laboratory centrifuge . 5 ) discard the supernatant taking care that the pellet is not dislodged . if the pellet size does not reduce appreciably ( to 1 / 10 th to 1 / 20 th of the original sputum volume ), give another wash with about 2 - 5 ml of solution 1 . 6 ) wash with sterile triple distilled water ( while resuspending the pellet in water , it should be done only by vortexing . optimal results may not be obtained if this is not followed .). the pellet is now ready for smear microscopy , culture , dna / rna isolation and pcr tests . 1 ) add an equal volume of solution 1 to the blood sample and mix . if the blood is partially clotted , incubate with solution 1 at 37 ° c . for half an hour . 2 ) pellet down the sample at medium ( 4000 - 5000 rpm , for sample volumes & gt ; 1 . 3 ml using 15 ml or 50 ml tubes ) or high ( 12 , 000 - 15 , 000 rpm , for sample volumes & lt ; 1 . 3 ml using 1 . 5 ml tubes ) speed . 3 ) if necessary wash the pellet once or twice with solution 1 till a buff coloured pellet is obtained . 4 ) wash the pellet with sterile triple distilled water . resuspend the pellet in water by vortexing and not by pipetting . ( optimal results may not be obtained if this is not followed .). 5 ) the pellet is now ready for smear microscopy , culture dna / rna isolation and pcr tests . processing of other body fluids ( except sputum and stored pleural fluid samnples ): 1 ) for sample volumes & gt ; 1 . 3 ml : pellet down the sample at medium speed ( 4000 - 5000 rpm ) in a 15 ml or 50 ml screw capped vial . 2 ) for sample volumes & lt ; 1 . 3 ml : pellet down the sample at high speed ( 12 , 000 - 15 , 000 rpm ) in a 1 . 5 ml polypropylene vial . 3 ) note : in case of very thick pus samples add equal to 1 1 / 2 volume of solution 1 to the sample and mix by vortexing , and add some solution 2 before pelleting . in case of bovine milk samples , remove the milk fat with a sterile cotton swab after centrifugation . 4 ) wash the pellet with 1 . 5 - 2 times pellet volume of solution lonce or twice followed by a wash with sterile triple distilled water . resuspend the pellet in water by vortexing and not by pipetting . ( optimal results may not be obtained if this is not followed .). the pellet is now ready for smear microscopy , culture dna / rna isolation and pcr tests . if the pleural fluid is processed within 1 hour after collection , then processing can be carried out as described in “ processing of other body fluids .” if the pleural effusion is stored for more than one hour the proteins separate out as a coagulum . such a sample should be processed as follows : 1 ) treat the coagulated protein ( obtained by decanting the supernatant into another tube ) separately with usp solution at 37 ° c . or room temperature for 30 minutes till it dissolves . 2 ) mix it with the supernatant and add some more solution 1 and about 10ml solution 2 and centrifuge at medium speed ( 4 , 000 - 5 , 000 rpm .) in a 15 ml or 50 ml screw capped vial . 3 ) if the pellet does not form properly add some more solution 2 and pellet again . 4 ) to the pellet give a second wash with solution 1 followed by water ( 10 - 15 ml ) wash . 5 ) the pellet is now ready for smear microscopy , culture dna / rna isolation and pcr tests . note : the pellet obtained from processing the liquid portion of the sample should be pooled with the processed pellet obtained from the coagulum . if the pleural fluid is collected in an edta vial , which prevents formation of coagulum , processing can be carried out as described in “ processing of other body fluids .” 1 ) add solution 1 to the vial containing the biopsy after removing the normal saline or the fluid in which it is contained , and transfer it to a sterile petri dish after 15 - 20 minutes .) this aids to the softening of the tissue material , which is necessary for the subsequent processing . 2 ) after the biopsy material becomes soft , mince it as finely as possible with sterile scalpel . the mincing should be fine to get optimal results . 3 ) transfer the minced biopsy material into a mini bead - beater vial ( 1 . 5 ml capacity ) containing at least half the volume of sterile 1 mm glass beads . the volume should not exceed 1 ml . 4 ) beat in a mini bead beater ( mini - beadbeater ™, biospec products , usa ) for 30 - 60 seconds . repeat bead beating once more for 30 seconds if the material is not homogenized properly . 5 ) centrifuge the vial at 2000 rpm for two minutes and collect the homogenate into a fresh vial avoiding collection of any glass beads . 6 ) centrifuge the homogenate at 12000 - 15000 rpm for 10 minutes at 25 ° c . and discard the supernatant . 7 ) wash the pellet once again with solution 1 ( 1 ml volume maximum ) followed by a wash with sterile water . 8 ) the pellet is now ready for smear microscopy , culture dna / rna isolation and pcr tests . note : for paraffin embedded tissue samples , deparaffinization with xylene is necessary before processing . to the pellet obtained after wash with sterile water , add 500 μl of solution 3 and resuspend the pellet thoroughly . it may be noted that the pellet has a tendency to sediment along the wall of the tube as well as at the bottom after centrifugation . care is to be taken that the pellet is completely resuspended to yield a homogeneous solution . this is important to obtain optimal results . transfer it to a 1 . 5 ml polypropylene vial . the material is now ready for the three tests namely , smear microscopy , culture and pcr . smear 50 μl on a clean glass slide in a minimum area possible . if there is any particulate matter in the smear , care should be taken to spread it out evenly with the inoculation loop while smearing . area of smearing preferably should not exceed 1 cm × 1 cm . simplified method of usp smear preparation : add 2 - 3 volumes of usp solution to the sputum ( specimen volume less than or equal to 2 ml ) collected in mccartney bottles . mix by hand for 30 - 60 seconds to homogenize well . allow to stand for 5 - 10 minutes . for highly tenacious and purulent sputum specimens , add at least 3 volumes of solution 1 , hand mix and incubate at 37 ° c . for 20 - 30 min . fill the bottle with sterile water up to ⅔ rd level and mix by inversion . centrifuge at 3 , 000 g for 20 minutes in an ambient temperature clinical centrifuge . decant supernatant carefully taking care that the pellet is not dislodged / lost . re - suspend the pellet in 2 - 5 ml of solution 1 and centrifuge as before . decant the supernatant carefully and wash the pellet with ˜ 10 ml sterile water . decant the supernatant carefully , add ˜ 10 drops of solution 2 with a dropper / pasteur pipet to the pellet and re - suspend it thoroughly with the help of an inoculation loop . take care to include in the final re - suspension the pellet deposited along the wall of the bottle during centrifugation . pour 34 drops of the re - suspension on a clean slide and spread using a continuous rotational movement with an inoculation loop to produce a smear of about 20 mm by 10 mm . allow the smear to air - dry for about 30 to 45 min , fix over flame , stain for afb using zn stain and examine slide under oil - immersion lens using a light microscope . c .) dna isolation for pcr ( 40 % of the resuspension is used ) for dna isolation , pellet the ‘ solution 3 resuspension ’ at 12 , 000 - 13 , 000 rpm in a microcentrifuge tube . to the pellet add approximately 5 times the volume of solution a , and 1 / 10 th volume of each of solution b and solution c , as that of solution a . mix well , avoiding frothing . heat at 90 ° c . for 40 minutes . centrifuge at room temperature at 12 , 000 rpm for 5 - 10 minutes . perform pcr with supernatant . be careful not to add any debris or resin from solution a into the pcr reaction . note : if the pellet size is drastically reduced so that it is not visible at all or the pellet size is very minute or the sample is a high bacillary load sample , lysis may be carried out by heating the ‘ solution 3 resuspension ’ directly or after adding triton x - 100 ( at a final concentration of 0 . 01 %) at 90 - 100 ° c . for 30 - 40 minutes . the supernatant may be used directly for pcr . the addition of the lysis reagents ( namely , solutions a , b and c ) may be avoided in such cases ; but for best results , it is recommended that the lysis reagents ( solution a , b and c ) be used for isolation of pcr - amplifiable dna ( see below for details ). d ). rna isolation : the pellet obtained can be subjected to mycobacterial rna isolation by any standard rna isolation procedures or kits . the pcr assay targets the devr gene ( rv3133c ) of m . tuberculosis and is specific for the organisms of the m . tuberculosis complex [ singh et al ., 1999 ]. in published studies from our laboratory , the primer pair devrf and devrr was used in the ‘ long target ’ pcr assay that amplified a 513 bp fragment from the devr gene [ singh et al ., 1999 ; 2000 ]. in a multi - centric study under dbt - funded project entitled “ evaluation of pcr - based test for tuberculosis ”, [ duration february 1997 - february 2000 , with financial support till 28 . 2 . 1999 ], the same pcr assay was used with coded sputum samples . the results of smear microscopy and culture were correlated with pcr results . sensitvity was 100 % for culture positive ( 14 / 14 culture positive ) and 94 % for smear positive samples ( 17 / 18 smear positive samples ) [ ref : dbt report on the analysis of the results of evaluation of different indigenously developed pcr assays for the diagnosis of tuberculosis . dbt , govt . of india ]. it was reasoned that amplification of a smaller region of the devr gene was likely to provide equivalent / better sensitivity with paucibacillary and smear negative samples . with this idea in mind two new primer pairs were designed to amplify shorter fragments of the devr gene . the ‘ short target ’ pcr assays were assessed first on purified m . tuberculosis dna and subsequently validated on clinical specimens . the ‘ short target ’ primer pairs are , ( i ) devrf2 , 5 ′ tggcaacggcattgaactgt 3 ′ and devrr2 , 5 ′ taagcaggcccagtagcgt 3 ′ and ( ii ) devrf3 , 5 ′ atctgttgtcccgcatgcc 3 ′ and devrr3 , 5 ′ gtccagc gcccacatcitt 3 ′. ‘ short target ’ assay : initial denaturation at 94 ° c . for 10 minutes ; 45 cycles of denaturation at 94 ° c . for 1 minute , annealing at 52 ° c . for 1 minute and extension at 72 ° c . for 30 seconds ; and a final extension at 72 ° c . for 10 minutes . ‘ long target ’ assay : identical parameters as for the ‘ short target ’ assay except that annealing and extension were performed at 65 ° c . for 90 seconds . is6110 assay : published primers suggested by eisenach et al . ( 1990 ) were used with the pcr reaction parameters : initial denaturation at 94 ° c . for 10 minutes followed by 45 cycles of 94 ° c . for 1 min and 60 ° c . for 1 min . 30 seconds , followed by a final extension at 72 ° c . for 5 minutes . cocktail components stock concentration working concentration 1 .) pcr reaction buffer 10 x 1 x 2 .) mgcl 2 25 mm / 50 mm 1 . 5 mm 3 .) primers 20 μm * 0 . 5 μm 4 .) dntps 10 mm 0 . 2 mm 5 .) taq polymerase 5 u / μl 1 u * to be reconstituted in pcr - grade water to obtain 20 μm concentration . total reaction volume is 40 μl ( 30 μl cocktail and 10 μl template ). dispense 30 μl cocktail into sterile 0 . 2 ml or 0 . 5 ml vials in a dna - free hood . add 10 μl template in a designated area away from the pcr - setup area . give the tubes a brief spin for 30 seconds and place them in the thermal cycler . after the completion of the reaction ( parameters given above ) electrophorese the amplification products in 1 . 5 - 2 . 3 % agarose gel in 0 . 5 × tbe buffer at 80 volts for 20 - 30 minutes . visualize the product ( load at least 35 μl ) by ethidium bromide staining under uv light . ( a ) function of usp solution components : to check for the function of each component of the usp solution , the usp solution was selectively depleted of the individual components and samples processed with the resulting solutions . usp solution consists of five components : 1 . guanidinium hydrochloride ( guhcl ) 2 . sarcosyl 3 . β - mercaptoethanol . 4 . tris - hcl 5 . edta it was apparent that the mucolytic , decontaminating , protein denaturing , chaotropic , liquefying , tissue softening / digesting , mycobacteria - releasing actions which are the principal functions of usp solution are performed by the first three components , namely guhcl , sarcosyl and β - mercaptoethanol . to establish this , these three components were selectively depleted from the usp solution individually or in combination keeping the tris - hcl and edta composition unchanged . the selectively depleted solutions were assessed for their effect on specimen processing , smear microscopy , pcr and culture ( fig1 to 4 ). a sputum specimen ( obtained by pooling three to four afb positive sputa ( to obtain a large volume and heterogeneous composition in terms of contar its ), was homogenized by vortexing with 3 mm glass beads for 3 - 5 minutes . aliquots of 5 ml each of the homogenized sputum were distributed for processing with the solutions of different compositions . usp solution was used as a control . the following solutions of varying compositions were prepared for sample processing : 1 . usp solution containing all the components ( called usp ). 2 . usp solution without guhcl ( called usp - 1 ). 3 . usp solution without sarcosyl ( called usp - 2 ). 4 . usp solution without β - mercaptoethanol ( called usp - 3 ) 5 . usp solution without guhcl and sarcosyl ( called usp - 1 , 2 ) 6 . usp solution without guhcl and β - mercaptoethanol ( called usp - 1 , 3 ) 7 . usp solution without guhcl , sarcosyl and β - mercaptoethanol ( called usp - 1 , 2 , 3 ) 8 . usp solution without sarcosyl and β - mercaptoethanol ( called usp - 2 , 3 ). all the solutions were used for processing of the sputum specimen as described and smear microscopy , pcr and culture was performed . the experiment was repeated twice for samples with varying physical characteristics . 1 . there was no reduction in pellet size when the sputum specimens were processed with usp solution lacking the three principal components ( usp - 1 , 2 , 3 , fig1 ). excessively thick smears with a lot of background were obtained when 10 % of the final processed pellet was smeared onto the slide , which was very difficult to read . culture contamination came within two days of inoculation on lj medium and pcr showed inhibition . 2 . whatever may be the variation of composition , the usp solution always fared the best in terms of junk removal and pcr inhibitor removal from the sample , producing the most clean pellet from the sputum specimen ( fig1 , fig3 ). 3 . for sputum specimens containing blood , guhcl acted as the principal inhibitor removal component of the sample by denaturing the hemoglobin and removing it from the sample . for specimens with high purulence and blood , pcr inhibition was noted in the dna preparations of samples processed with usp solution not containing guhcl . 4 . it was seen that in case of mucoid sputum samples , usp solution that contained only sarcosyl and β - mercaptoethanol [ i . e . without guhcl ( usp - 1 )], effectively processed the sample with commendable efficiency and better than usp containing only guhcl ( usp - 2 , 3 ) as far as smear microscopy and pcr were concerned ( fig1 and 3 ). however , usp - 1 failed to adequately process ( in terms of efficiency of junk removal and inhibitor removal ) sputum specimens of high purulent nature . in such cases usp solution containing only guhcl ( usp - 2 , 3 ) or guhcl with sarcosyl ( usp - 3 ) fared better . so it was evident that the presence of guhcl and sarcosyl in usp was more suitable for purulent samples and the presence of β - mercaptoethanol and sarcosyl was more suitable for mucoid samples . though even for mucoid samples , the presence of guhcl was necessary to remove pcr inhibition efficiently . but for mucopurulent samples the presence of guhcl along with β - mercaptoethanol and / or sarcosyl was absolutely necessary for optimal processing . 5 . whenever guhcl was subtracted from the usp solution , the resulting cultures showed contamination when inoculated on lj medium ( fig4 a ). presence of guhcl in the solution was necessary for proper decontamination of the specimens . 6 . depletion of any one of the principal components resulted in increased background in the smears when smears were prepared by using 10 % of the fmal processed pellet ( fig2 ), which interferes with ease in slide reading which is a major advantage of the usp method ( see following sections ). 7 . thus it was evident that the three principal components in the usp solution act in a synergistic manner to produce the optimal effect in processing the sample and the absence of any one of these components results in sub - optimal results . the versatility of the usp solution in processing specimens of a wide range of physical characteristics and biochemical nature with equal efficiency can be attributed to the synergistic effect of all the components of the usp solution . tris - hcl helps in maintaining the neutral ph of the usp solution and thus there is no need for neutralization of the usp solution - treated specimen before culturing . edta acts as a chelator of positive ions , thereby inactivating dnase . guhcl is a potent chaotrope thereby helping in denaturing all the host proteins in the specimen including mucin and hemoglobin thereby removing potential pcr inhibitors and counterstaining material from the clinical specimen . it kills gram - negative bacteria present in the specimen thereby decontaminating it ( see below ). it also denatures dnases and rnases thereby preserving nucleic acids and making the procedure amenable to dna and rna isolation . sarcosyl solubilizes lipids and disrupts cell membranes of eukaryotic cells and gram negative bacteria and helps in decontaminating the sample and releasing mycobacteria from macrophages . β - mercaptoethanol , a reducing agent acts as a mucolytic component reducing the s — s bonds of mucin to — sh groups and releases mycobacteria trapped in the mucous . the tough cell wall characteristics of mycobacteria makes it totally inert to any adverse effect by the usp solution and thus the method is eminently suitable for processing samples as a prelude to mycobacterial diagnostics by smear microscopy , culture , pcr and rt - pcr ( for rna ). ( b ) rapid liquefying activity of usp . fig5 a and fig5 b show representative sputum and tissue samples treated with usp . at the end of the process the sample is rid of contaminating organisms , proteins , enzymes and interfering substances and considerably reduced in volume . ( c ) smear microscopy . representative slide smears prepared by the usp , cdc and direct smear methods are shown in fig6 . the appreciable decrease in background staining material is noteworthy in the usp slide . ( i ) sensitivity of the usp method of smear microscopy . m . tuberculosis h37rv was grown up to logarithmic phase in middlebrook 7h9 medium containing adc and 0 . 05 % tween 80 . the culture was serially diluted in duplicate sets as follows : 1 : 10 , 1 : 100 , 1 : 1000 and so on till 1 : 100000 . 10 % of each dilution was inoculated on lj slants ( solid medium ) and 100 % of each of the dilution the other set was spiked into 1 ml sputum aliquots from a patient suffering from chronic obstructive pulmonary disease ( copd ) without any history or symptoms of tb . the spiked samples were processed by the usp method and 10 % of each sample was smeared on a glass slide , stained for afb and microscopically examined under oil immersion lens . a similar experiment was performed using copd sputum samples spiked with m . smegniatis , a non - pathogenic fast growing mycobacterium . from both the experiments it was established that the usp method of smear microscopy could detect as positive , samples that contained 300 - 400 afb / ml of the sample . the results were verified twice more . a representative experiment is shown in fig7 . these results clearly illustrate the superior sensitivity of the usp method of smear microscopy . a ˜ 30 fold enhancement in sensitivity over the conventional direct smear microscopy method was noted . the sensitivity of detection of the direct method is ˜ 10 , 000 bacilli / ml of sample ( kent and kubica , 1995 ). ( ii ) evaluation of usp smear microscopy in a cilincal setting . the performance of usp smear microscopy was evaluated on a panel of 571 sputa collected from patients ( n = 571 ) attending the dots centre at new delhi tuberculosis centre , new delhi ; sunderlal jain charitable trust hospital , delhi and tuberculosis research centre , chennai for diagnosis of pulmonary tuberculosis . the patients chosen for this study were evaluated for any of the following clinical symptoms namely , fever , cough , expectoration of sputum , haemoptysis , pain , dyspnoea and other symptoms like loss in weight , night sweats and general weakness , x - ray chest , mantoux status and any past history of tuberculosis . any specimen collected from a subject already on att at the time of specimen collection was excluded from the study . culture results on lj media was used as a gold standard . all the specimens were coded and the study was blinded . ( iii ) the performance of usp riethod was evaluated with respect to both the direct smear and the nalc — naoh concentration methods , the two most universally applied methods of smear microscopy . all 571 samples were processed for smear microscopy by the direct method and the usp method ( data set 1 ). out of these , 325 samples ( data set 2 ) were also processed by the nalc — naoh concentration method ( developed at cdc , atlanta and called cdc method ). the usp and cdc methods were performed on what were considered to be approximately equal aliquots of the sputum samples . the direct smears were independently prepared and read at two different sites by different trained personnel . the smears prepared by the usp and cdc methods were read by at least two trained personnel at one laboratory . the improvements in usp smear method over the direct smear method and the cdc method are : a ). negative samples turn to positive in usp smear : the sensitivity of the direct smear method and the usp smear method were 68 . 6 % and 98 . 2 %, respectively , with a specificity of 92 . 6 % and 91 . 4 % respectively on the panel of 571 sputum sample ( data set 1 ). all samples diagnosed as smear positive by the direct method were also positive by the usp and cdc methods of smear microscopy . out of the 325 direct smear - negative samples , the usp method detected 97 additional samples as smear positive , which were positive by culture also , thereby recording an enhancement in sensitivity of 29 . 6 %. a representative example is shown in fig8 . out of the 97 usp smear - positive samples , 14 were graded as scanty , 29 as 1 +, 23 as 2 + and 31 as 3 +. the usp smear method was also simultaneously compared with cdc method of smear microscopy in 325 out of these 571 specimens ( data set 2 ). 29 specimens , which were smear negative by the cdc method , were detected as positive by the usp method of smear microscopy all of which were also culture positive . thus usp method also fared better than a popularly used concentration method of smear microscopy ( cdc method ) in terms of sensitivity of detection ( 97 . 1 % and 80 % sensitivity for usp and cdc smear microscopy , respectively ). culture smear method and result positive negative usp any positive 322 21 negative 6 222 total 328 243 direct any positive 225 18 negative 103 225 total 328 243 culture smear method and result positive negative usp any positive 165 26 negative 5 129 total 170 155 cdc any positive 136 16 negative 34 139 total 170 155 b ). udgradation in smear status : the usp method enhances the gradation of slides making them easier to read in a shorter period of time . slides graded as scanty , 1 + or 2 + by the direct method of smear microscopy generally turned into 1 +, 2 + or 3 + by the usp method . this trend was observed when the results of usp method of smear microscopy were compared with that of the direct method over the panel of 571 samples ( table 1 ). the upgradation in the smear status was also observed when the usp solution method was compared with the cdc method ( table 2 ). representative slides are shown in fig9 . thus the usp method also fared better than the cdc method in terms of the number of bacilli observable per field . this facilitated rapid and efficient reading of the slides ultimately resulting in saving of the technician &# 39 ; s time and labour . thus the usp solution method of smearing showed a very high sensitivity in the range of 97 - 98 %, much better than the other two methods that were tested , with a specificity range of 83 - 91 %. usp smear microscopy showed a positivity of 97 - 98 % in culture positive samples vs . 80 % by cdc and 68 . 6 % by direct smear methods thereby recording an enhancement in sensitivity of ˜ 30 % and 18 % over the direct and cdc method of smear microscopy respectively . the increase in the sensitivity was significant as the sensitivity values of all the three methods were completely non - verlapping at a 95 % confidence interval . the usp smear method did not miss a single sample detected by either the direct smear method or the cdc method . the specificity of the usp smear microscopy method when compared to the direct smear method was 91 . 3 % and that when compared to the cdc method was 83 . 3 %. the two values were overlapping at a 95 % confidence interval . ( data sets 1 and 2 ). ( i ) lethality of usp solution on m . tuberculosis spiked in sputum . a logarithmic phase m . tuberculosis culture was serially diluted up to 10000 - fold in duplicate . 10 % of one set of dilutions was inoculated onto lj slant , 100 % of each dilution of the duplicate set was spiked into 1 ml of sputum aliquots obtained from a patient suffering from copd . the spiked samples were processed by the usp method and 10 % of the processed material was inoculated onto lj slant . the viable count obtained in the case of untreated cells was 720 / ml and that in case of the spiked usp solution treated cells was 400 / ml ( at 10000 - fold dilution ) after 8 weeks ( table 3 ; fig1 ). approximately 55 % of mycobacteria survived treatment with usp . the cdc method that is currently the most popularly used method for decontamination of clinical samples employs naoh and with this method , a loss of viability from 28 to 70 % has been reported [ krasnow and wayne . 1966 ]. however it was found that usp solution containing 4 m guhcl ( in place of 5 m guhcl ) was less harsh on mycobacteria . more viable bacterial colonies were obtained when m . tuberculosis was treated with usp containing 4 m guhcl than with usp having 6m guhcl ); 142 cells / ml in case of cells treated with usp containing 4 m guhcl and 72 cells / ml in case of cells treated with 6 m guhcl ( at 100 fold dilution ) after 4 weeks . the untreated cells of the same dilution corresponded to 277 cells / ml . but it decontaminated clinical samples with equal efficiency . so where culturing of m . tuberculosis from clinical samples is of prime importance , usp solution containing 4m guhcl will increase the chances of culture positivity . ( ii ) effect of usp on the growth rate of m . tuberculosis : it is well known that treatment with decontaminating reagents like naoh slows down the growth rate of m . tuberculosis from clinical material . a similar observation was made with usp solution - treated bacteria also . usp - treated m . tuberculosis colonies appeared after a lag of at least 1 - 1 1 / 2 weeks compared to that of untreated cells on solid media ( middlebrook 7h10 or lj ). nalc — naoh treated m . tuberculosis ( cdc method ) also grew somewhat faster ( visible colonies were obtained at least 45 days earlier ) than usp - treated bacteria on solid media although no appreciable differences were observed between the two treatments when bacteria were cultured in liquid system bd bactec ™ mgit ™ 960 system ( becton dickinson , usa ) [ see below ]. from the above experiments it was estimated that after usp ( containing 5 m guhcl ) solution treatment at 37 ° c . for 20 minutes , 56 - 62 % of the m . tuberculosis cells were rendered non - viable . thus , usp solution is not grossly lethal to the m . tuberculosis . ( ili ). the growth rate of m . tuberculosis is equivalent after treatment with usp solution and nalc — naoh in liquid medium ( mycobacterial growth indicator tubes ): it is well known that treatment with decontaminating agents adversely affects the viability of mycobacteria and slows down their growth rate . the effect of usp solution treatment on the growth rate of m . tuberculosis was compared to that of nalc — naoh treatment ( cdc method ). a logarithmic phase m . tuberculosis culture grown in middlebrook 7h9 liquid medium ( with adc supplement and 0 . 05 % tween 80 ) was divided into three sets . one set was treated with usp solution and the other set was treated with nalc — naoh followed by neutralization with phosphate buffer ( cdc protocol ). all three sets ( i . e ., usp solution - treated , nalc — naoh - treated and untreated ) were inoculated into mgit tubes with panta reagent and oadc supplement ( becton dickinson , usa ), and monitored for positive signal using bd bactec ™ mgit ™ 960 system . untreated cells gave a positive signal for growth in two days whereas the usp solution - treated cells and nalc — naoh - treated cells gave a positive signal for growth in five days . this experiment was repeated and the same results were obtained . this suggests that both usp and cdc methods slow down the growth of m . tuberculosis in liquid medium ( mgm ) to an equal extent . ( iv ) evaluation of usp culture in a clinical setting . the usp method of culturing was compared with other conventional methods of culturing ( cdc method , modified pettrof &# 39 ; s method and nassua ‘ s method ) on a panel of 571 specimens . ( a ). both the usp and the conventional methods showed comparable percent - positivity . the positivity of the conventional methods ( 53 . 6 %) was not significantly higher than that of the usp method ( 50 . 1 %) and they overlapped at 95 % confidence interval . ( b ). usp method proved to be very efficient in decontamination and showed lesser number of contaminated cultures ( n = 5 ) compared to the conventional methods ( n = 21 ) thereby proving it to be more suitable for tropical countries . ( c ). usp solution being a neutral ph solution , samples decontaminated with usp solution did not require any neutralization prior to culturing . thus in terms of sensitivity , effect on viability and effect on growth rate , the usp method of culture is comparable to the currently accepted methods for the culturing of m . tuberculosis from clinical samples . the pcr test was performed on & gt ; 700 samples of pulmonary and extra - pulmornary origin . samples were either fresh or frozen at − 20 ° c . for up to 2 months . mycobacterial dna was isolated for this purpose by the usp method . not a single sample has showed inhibition in the pcr assay indicating that the dna isolated from the clinical samples by the usp method was free of inhibitors . the pcr assay targets the devr gene of m . tuberculosis ( rv 3133c ) and is specific for the organisms of the m . tuberculosis complex [ singh et al ., 1999 ]. in the published studies , the pcr primer pair devrf and devrr amplified a 513 bp fragment from the target gene [ singh et al ., 1999 ; 2000 ]. it was reasoned that amplification of a smaller region of the same gene was likely to provide equivalent / better sensitivity with paucibacillary and smear negative samples . with this idea in mind two new sets of primers mapping within the devr gene were used in order to amplify shorter fragments of the devr gene . the new primer pairs used were ( i ) devrf2 and devrr2 that amplify a 308 bp fragment ( fig1 ) and ( ii ) devrf and devrr3 that amplify a 164 bp , respectively from the devr gene ( fig1 ). the results were compared with the original 513 bp devr assay and an is6110 - based assay which is used in a large number of laboratories worldwide . ( i ) assay specificity . the specificity of the assays was assessed by the addition of dna from various mycobacterial species into the pcr reaction . the devrf2 and devrr2 primers showed excellent specificity and a band of expected size ( 308 bp ), was obtained only with dna from organisms belonging to the m . tuberculosis complex and not with dna from other mycobacterial species ( fig1 ). however , with devrf3 and devrr3 primers , amplification ( 164 bp product ) was obtained with dna from m . kansasii and m . xenopi species in addition to m . africanum , m . bovis and m . tuberculosis . further , no amplification was obtained with dna from m . microti , which belongs to the m . tuberculosis complex ( fig1 ). ( ii ) assay sensitivity using purified dna . serial dilutions of pure m . tuberculosis dna were used in pcr assays to assess the limit of detection of the amplified products by ethidium bromide ( table 4 , fig1 ). in comparison to the pcr assay that generated a 513 bp amplification product , the primer pairs devrf2 - devrr2 and devrf3 - devrr3 showed a marked enhancement in sensitivity . the devrf2 and devrr2 primers [ product size 308 bp ] showed 2 - to 4 - fold more sensitivity and the devrf3 and devrr3 primers ( product size , 164 bp ) showed at least 10 - fold more sensitivity over devrf and devrr primers ( product size , 513 bp ) [ table 4 ]. ( iii ) evaluation of the ‘ short target ’ and ‘ long target ’ pcr assays in a clinical setting . the results of the pcr assays were compared with that of culture and smear microscopy , of 571 samples collected from suspected tuberculosis patients ( n = 571 ) coming to the dots centre at new delhi tuberculosis center , sunderlal jain hospital , delhi and tuberculosis research center , chennai . the assays used were : 1 . ‘ short target ’ devr pcr assay using a combination of primer pairs devrf2 & amp ; devrr2 ( product size , 308 bp and devrf3 & amp ; devrr3 ( product size , 164 bp )( carried out on 571 sputum specimens ). 2 . ‘ long target ’ devr pcr assay using previously published and validated primer pairs devrf and devrr ( product size 513 bp ) [ singh et al 1998 , 2000 ] ( carried out on 506 of the 571 specimens ). 3 . is6110 pcr assay carried out on 571 samples using published primers eisenach et al 1990 ]. this assay served as a control since it is the most widely used pcr assay worldwide . the sensitivity of the ‘ short target ’ assay was 98 . 5 %. this was substantially better than that of the ‘ long target ’ assay , whose sensitivity was 73 . 2 %. for example , out of the 571 sputum samples analyzed , 322 sputum samples were positive by the usp smear method and also culture positive . out of these culture positive and usp smear positive samples , long target assay failed to detect 65 samples , which were detected , by the short target assay confirming the latter assay to be a more sensitive one . thus the introduction of the ‘ short target ’ primers increased the devr assay sensitivity by 25 . 3 % using culture positivity , as gold standard ( data set 3 ) and 2 - 10 folds when compared using serial dilutions of m . tuberculosis genomic dna ( table 4 ). the overall diagnostic accuracy of the short - target assay was about 7 % higher than that of the long - target assay ( data set 3 ). the enhanced sensitivity of the ‘ short target ’ assay was however accompanied by a lower specificity of 77 % as compared to a specificity of 91 . 9 % with the ‘ long target ’ assay ( data set 3 ). the apparent reduction in specificity of the ‘ short target ’ assay is likely a reflection of the enhanced sensitivity of the assay and is unlikely to be due to false positivity / lower specificity . this is supported by the following : ( i ) out of 328 culture positive samples , the ‘ short target ’ pcr detected 323 samples . an equal number were also positive by is6110 pcr assay . however in this study covering 571 sarnples , 6 samples were negative by the is6110 assay while being positive for the devr ‘ short target ’ assay probably due to absence of is6110 gene . the is6110 pcr also detected 2 additional culture positive samples ( data set 3 ). the is6110 - based pcr assay for amplification of a 123 bp product is specific for organisms of m . tuberculosis complex and shows high sensitivity on account of the presence of is6110 in multiple copies in the genome ( as opposed to devr which is present in a single copy per genome ). but the devr short target assay showed comparable sensitivity to is6110 assay ( data set 3 ). the value of devr also lies in its universal presence in all m . tuberculosis isolates in comparison to is6110 that is reported to be sometimes completely absent or to be present in 1 or few copies in indian strains ( narayanan et al 2001 ). this is reflected in the lower sensitivity of the is6110 assay in the case of pleural tissue and lymph node specimens in the study with extrapulmonary specimens described later ( data sets 5 & amp ; 6 ). ( ii ) non - tuberculosis controls : smear microscopy , culture and pcr were carried out on a panel of 78 sputum from 69 patients suffering from diseases clinically proven to be other than tuberculosis . all the samples were negative by smear microscopy , pcr and culture proving the specificity of the assay systems . sensitivity and specificity of pcr using ‘ long target ’ and ‘ short target ’ devr pcr ). using culture positivity as gold standard ; sample strength , n = 506 for devr - long pcr n = 571 for devr - short pcr . culture pcr target and result positive negative devr - long ( 513 bp ) positive 199 19 negative 73 215 total 272 234 devr - short ( 308 / 164 bp ) positive 323 56 negative 5 187 total 328 243 is6110 ( 123 bp ) positive 325 70 negative 3 173 total 328 243 extrapulmonary samples : the usp method was evaluated for its performance in the diagnosis of tuberculous pleural effusion and lymphadenopathy in a blinded study with coded pleural fluid , pleural tissue and lymph - node biopsy specimens collected from the patients coming to the opd of safdaijung hospital , new delhi , india . the samples comprised of 100 specimens from 88 patients including 77 specimens from tb patients and 23 specimens from control subjects with diseases like malignancy , arnoebiasis , chf , sarcoidosis and reactive lymphadenopathy ( nonspecific inflammation ). inclusion criteria was based on any one or more of the following parameters : a ) histopathology / cytopathology consistent with tb ( b ) positive smear for afb ( c ) culture positive for m . tb . ( d ) clinical response to att . specimens were processed by the usp method and smear microscopy , culture and pcr were performed . in this study a comparison between devr and is6110 based pcr was carried out . devr pcr was carried out with the primer pairs devrf3 / devrr3 m . tuberculosis dna that was isolated from the specimens was of good quality and no pcr inhibition was noticeable . the usp method of smear microscopy detected eight specimens ( 6 pleural fluid and one each of lymph node and pleural tissue biopsy ) ( data set 4 ) as positive against three with the conventional method of smear microscopy . the sensitivity of usp smear microscopy was 10 . 4 % in this group of extrapulmonary specimens whereas that of the conventional method was only 3 . 9 %. thus the usp methods enabled improved conclusive afb smear microscopy from tissue biopsy specimens and other extrapulmonary specimens , compared . to routine diagnostic methods . five specimens were positive by the usp method of culturing ( sensitivity 6 . 5 %)( data set 4 ). the devr pcr showed better sensitivity with pleural tissue and lymph node ( 100 % and 75 % respectively ) specimens than is6110 , ( 75 % and 50 % respectively ) probably due to absence of any copies of is6110 in these strains ( data sets 5 & amp ; 6 ). thus the usp method of specimen processing and devr pcr served as a useful modality to arrive at the definitive diagnosis of tuberculous pleural effusion and lymphadenopathy . smear microscopy and culture status of extrapulmonary specimens processed by the usp method sensitivity and specificity values (%) for devr - short target pcr for the extrapulmonary samples sensitivity and specificity values (%) for is6110 pcr for the extrapulmonary samples . pleural fluid pleural tissue lymph node sensitivity (%) 93 75 50 specificity (%) 96 . 15 100 75 ppv (%) 97 . 6 100 88 . 9 npv (%) 89 . 3 60 27 . 3 efficiency (%) 94 . 2 81 . 9 55 the statistical analysis was done using sas 8 . 0 software ( sas institute inc . cary , n . c ., usa .). descriptive statistics , frequency distribution and percentage of the variables have been calculated . to evaluate diagnostic accuracy and reliability of the tests in comparison to gold standards , sensitivity , specificity , positive and negative predictive values and their confidence intervals at 95 % have been calculated . ( f ) simplified protocol for lysis of mycobacteria present in the processed specimen prior to dna isolation for pcr : after usp processing if the pellet size is drastically reduced so as to be nearly invisible or is very minute or if the sample is a high bacillary load sample , lysis may be carried out by heating the resuspension directly in solution 3 or after addition of triton x - 100 ( final concentration of 0 . 01 %) at 90 - 100 ° c . for 30 - 40 minutes . three afb positive sputum specimens were processed by the usp method . the processed pellet was resuspended into resuspension solution ( solution 3 ) and divided into three equal aliquots for each specimen . one aliquot was pelleted and to the pellet was added lysis reagents ( namely , solutions a , b and c ), to the second aliquot was added triton x - 100 at a final concentration of 0 . 01 % and the other was kept as such . all the three were boiled at 90 ° c . for 40 minutes and pcr was carried out with the supernatant . all the three lysates in each of the three specimens yielded amplicons with almost equal intensity when analysed by agarose gel electrophoresis ( fig1 ). these results indicate that in case of very clean pellet obtained after processing or for samples with high bacillary loads , the addition of the lysis reagents ( namely , solutions a , b and c ) may be avoided ; but for best results , it is recommended that the lysis reagents ( solution a , b and c ) be used for isolation of pcr - amplifiable dna . ( g ) usp method is applicable to diagnosis of mycobacteriosis caused by mycobacterium other than tuberculosis type ( mott ) as it does not have any detrimental effect on them : motit bacilli were subjected to usp treatment for 10 - 15 minutes at room temperature , followed by a wash with sterile triple distilled water . each species was then subjected to smear microscopy , culture on lj slant and pcr to check for detrimental effect of usp solution , if any . the moct species used were : m . avium , m . fortuitum , m . gordonae , m . intracellulare , m . kansasi , m . scrofulacem , m . smegmatis , m . phlei , and m . vaccae . organisms belonging to the m . tuberculosis complex like m . aficatium , m . bovis , m . bovis ( bcg ), m . microti were also included in the study . all the organisms retained their afb status ( fig1 ) and viability . however m . smegmatis though it maintained its integrity and afb property , lost its viability . fig1 shows two usp - treated rapid growers . good quality pcr - amplifiable dna was isolated from all the usp - treated mycobacterium ( fig1 ). ( h ) samples processed by the usp method are compatible with procedures for rna isolation from m . tuberculosis : the usp method is also compatible with the isolation of high quality m . tuberculosis rna from sputum specimens . for this purpose usp solution with the following composition was used . 6m guanidinium hydrochloride , 50 mm tris - ci , ph 7 . 5 , 25 mm edta , 0 . 5 % sarcosyl and 0 . 2 m β - mercaptoethanol . the sputum specimen used was graded 3 + by the direct method of smear microscopy . to the specimen ( 5 - 7 ml ), was added 1 . 5 times the volume of usp solution and the mixture was homogenized . to it was added 10 ml of sterile depc water and mixed . it was then centrifuged at 5 , 000 rpm for 10 minutes at room temperature . the pellet was again washed with 2 - 3 ml of usp solution followed by a wash with sterile depc water . the final processed pellet can be used as a starting material for rna isolation by an appropriate means . for example , using the qiagen rneasy mini kit as per manufacturer ‘ s protocol . the isolated m . tuberculosis rna was subjected to reverse transcription reaction using stratrascript reverse transcriptase ( rt ) enzyme ( stratagene , usa ) to obtain cdna . the cdna was subjected to amplification using m . tuberculosis rrna -( 23s rrna ) and mrna ( rv 3134c gene )- specific primers to detect the presence of m . tuberculosis rna . rt negative controls in which the isolated rna was subjected to reverse transcription reaction without the reverse transcriptase enzyme were also run for pcr to monitor for the presence of any contaminating genomic dna in the preparation . amplifications corresponding to both the ribosomal and messenger cdnas were obtained ( fig1 ) proving that both classes of m . tuberculosis rna was isolated from the sputum specimen . ( 1 ) effect of usp solution on the viability and staining property of gram staining bacteria : the effect of usp solution on the viability and staining properties of escherichia coli , pseudomanas sp ., and staphylococcus sp ., the three most common microflora present in sputum specimens , was evaluated . the bacteria were treated with solution 1 for 10 - 15 minutes at room temperature followed by centrifugation and washing of the pellet with sterile water as described . the pellets were then resuspended in solution 3 and 10 % was smeared in glass slides ( in duplicates ) and 40 % was inoculated in nutrient agar media and incubated at 37 ° c . to monitor for any growth . this was followed by gram staining of the slides . gram staining was also performed on the untreated bacteria . it was observed that both pseudomonas sp . and e . coli , were lysed by treatment with usp solution as evident from the total loss of morphology as seen by gram staining ( fig2 ) and absence of any growth in the nutrient agar media even after one week . however staphylococcus sp . retained its morphology as well as viability ( checked by growth on nutrient - agar medium ) on treatment with usp solution . however it did not grow in lj media possibly due to selectivity of the media when a sputum sample containing both m . tuberculosis and staphylococcus sp . was processed by the usp method and cultured on lj media ( not shown ). so usp solution lyses and kills gram negative bacteria but does not have any detrimental effect on gram positive bacteria like staphylococcus sp . and hence can also be used for diagnosis of staphylococcal disease . ( i ) a methodology is developed that involves optimized sample processing using a novel use of a chaotropic agent to provide rapid , sensitive and accurate diagnosis of tuberculosis . the unique properties of the mycobacterial cell wall render mycobacteria selectively resistant to the action of guanidinium hydrochloride . ( ii ) in addition to m . tuberculosis and m . bovis , samples containing other mycobacteria can also be effectively processed by the usp method prior to subjecting the material to diagnostic tests . ( iii ) the claimed technology is modular ; it is eminently suited for highly sensitive smear microscopy , culture and isolation of good quality inhibitor - free mycobacterial dna and rna for use in nucleic acid - based diagnostic assays . further , a sensitive pcr assay is described that enables sensitive and specific diagnosis of tuberculosis . ( iv ) the methodology being relatively simple and user - friendly can be performed by technicians having minimal scientific background and training . 1 . the major advantage of the usp method over the existing methods of smear microscopy is its very high sensitivity . the method has been shown to reproducibly detect samples containing up to 300 - 400 bacilli / ml of sputum . this is ˜ 30 fold more sensitive than the direct method of smear examination . the sensitivity ( limit of detection ) can be further increased by using more than the prescribed 10 % of the processed specimen for smear microscopy . 2 . since the method deals with the whole sputum sample , samples that lack purulence or contain nasopharyngeal discharge or saliva can also be processed easily by this method . 3 . since tissue and cell debris and the proteinaceous waste in the sputum are effectively removed , the smear can be made in a minimal area ( 1 cm × 1 cm ). up to 10 % of the sample may be used with no chance of getting an excessively thick smear or obtaining a high background that interferes with the microscopic examination of the slides ( see fig2 ). 4 . in case culture and pcr tests are not performed , & gt ; 10 % of the sample can be examined on multiple slides . this approach is expected to further improve the sensitivity of smear microscopy by usp method . 5 . this method is particularly beneficial while preparing smears from tissue biopsy samples . it can bypass routine histopathological procedures for preparing smears from solid biopsy specimens for apb smear microscopy . this method yields high quality smears from tissue biopsy samples suitable for afb staining , which is not possible either by the direct or concentration ( cdc ) methods of smearing . 6 . the method is also compatible with mycobacteria other than m . tuberculosis . 1 . the process does not involve the use of costly reagents like nalc . 2 . the usp solution is a buffered solution having neutral ph . so neutralization of the sample is not necessary before culturing . 3 . the process is versatile and can be used for culturing m . tuberculosis from a wide range of pulmonary and extra - pulmonary samples in addition to sputum . 4 . the usp solution serves as a very effective decontaminant and is suitable for use in tropical countries with humid climate . 5 . the method is also compatible with culture of mycobacteria other than m . tuberculosis and gram positive organisms such as staphylococcus sp . 1 . this method being a multipurpose method , a separate method is not required for isolating dna / rna from the samples processed for smear microscopy and culture . 2 . it can isolate inhibitor - free pcr - amplifiable dna from a wide range of samples of pulmonary and extra - pulmonary origin , in fact it is claimed to be a universal processing method . it efficiently removes hemoglobin from clinical samples ( eg . blood , hemoptyic sputum samples ) which is known to be a potent pcr inhibitor ( mahony et al . 1998 ; al - soud and radstrom p 2001 ; kang et al . 2002 ). 3 . it does not involve the use of hazardous organic solvents like phenol or chloroform or expensive enzymes like lysozyme , etc . 4 . since it contains chaotropic agent guhcl , a potent rnase denaturant , it is compatible with high quality mycobacterial rna isolation from clinical specimens . 1 . the devr gene is conserved amongst all species and isolates of m . tuberculosis complex including m . tuberculosis and m . bovis in contrast to a popular assay based on insertion sequence is6110 . is6110 sequences have been reported to be missing in some proportion of clinical isolates of india and worldwide . [ dale et al . 1997 , barlow et al . 2001 , narayanan et al . 2001 ]. this technology is suitable for any kind of body fluid and tissue biopsy ( except for fecal matter which has not been tested ). the claimed technology can be used in a variety of clinical settings and laboratories . ( 1 ) it can be used for smear microscopy in laboratories having minimal equipment ( only a vortex shaker , light microscope and ambient temperature centrifuge that can go up to a speed of 4000 rpm ). ( 2 ) it can be used for smear microscopy and culture in laboratories having additional facilities for mycobacterial culture . ( 3 ) finally pcr and rt - pcr tests can be applied in sophisticated laboratories . the technology thus can be marketed - as the following to suit individual requirements : kit 1 : universal sample processing ( tjsp ) kit [ containing 46 m guhcl and 0 . 1 - 0 . 2 β - mercaptoethanol ; see “ detailed description ” for details ]. kit 4 : complete kit ( usp , smear , culture , dna isolation and pcr ). kit 5 : as a part of mycobacterial rna isolation kit from clinical specimens . 1 . this method alone serves all the three important aspects of tb diagnosis by molecular and microbiological methods , namely smear microscopy , culture and pcr / rt - pcr . in fact it is claimed to be a universal processing method . 2 . the methodology is versatile and applicable on all types of human clinical material including respiratory specimens ( spontaneously expectorated sputum , normal saline - nebulized induced sputum , transtracheal aspirate , bronchoalveolar lavage , laryngeal swab , nasopharyngeal swab ), body fluids ( pleural fluid , pericardial fluid , joint aspirate , gastric aspirate , peritoneal fluid , cerebrospinal fluid , urine , pus , endometrial aspirate , synovial fluid ), body tissues ( blood , bone marrow biopsy ) and solid organs ( lymph node , bone , skin ) and bovine samples ( lymph gland , milk and blood ). 3 . the method is rapid ; it requires a maximum of 2 hours for complete processing of a sample . 4 . the method is also suitable for smear microscopy , culture and pcr from frozen sputum samples ( stored at − 20 ° c . for up to 2 months ) and frozen extra pulmonary samples . 5 . the method is suitable for the isolation of host ( human ). dnta with certain modifications . 1 . safe : ( i ) usp solution contains guanidinium hydrochloride ( guhcl ) instead of guanidinium isothiocyanate ( guscn ) as the principal component . since guscn yields a potentially toxic gas hcn on contact with acids , extreme care is to be taken while disposing off solutions containing this chemical . therefore it is not suitable for unspecialized and routine microbiological laboratories . but the use of guhcl in this reagent makes it safe to handle and dispose . ( ii ) it does not use any hazardous organic solvents . 2 . stability and handing : no special handling required . 3 . cost effective : the usp method does not involve the use of the expensive reagent nalc unlike the irs , method . further , guhcl costs lesser than guscn which is a part of inhibitor removal solution . 4 . usp solution is a neutral ph solution : the usp solution is a buffered solution of a neutral ph . therefore no additional step involving neutralization is necessary before culturing of m . tuberculosis and other mycobacteria from the decontaminated samples . usp solution is also compatible with rna isolation protocols . 5 . mucolytic , decontaminating , protein denaturing , chaotropic , liquefying , tissue softeningldigesting , mycobacteria - releasing action . 6 . synergistic action of the three main components of usp : including chaotrope guhcl , detergent sarcosyl and reducing agent β - mercaptoethanol . 1 . the method is eminently suitable for the preparation of smears for highly sensitive microscopy . the method has been shown to reproducibly detect samples containing up to 300 - 400 bacilli / ml of sputum . this is ˜ 30 fold more sensitive than the direct method of smear examination . the sensitivity ( limit of detection ) can be further increased by using more than the prescribed 10 % of the processed specimen for smear microscopy . 2 . since the method deals with the whole sputum sample , samples that lack purulence or contain nasopharyngeal discharge or saliva can also be processed easily by this method . 3 . since this method effectively removes the tissue and cell debris and the proteinaceous waste in the sputum ( counterstaining material ), the smear can be made in a minimal area ( 1 cm × 1 cm ) taking at least 10 % of the sample with no chances of getting an excessively thick smear or obtaining a high background which interferes with the microscopic examination of the slides ( see fig9 b ). this enables more bacilli to be smeared on the slide thereby increasing the sensitivity of the microscopic detection method considerably and minimizing the time spent in examining every slide . thus the usp method generally converts slides that are graded as 1 + or scanty by the direct method to 2 +/ 3 + or 2 +/ 1 +, respectively . following the same principle , the slides that are read as negative by the direct method become positive by the usp method . it must be mentioned here that the number of fields scanned in case of negative samples for each method of smearing was 300 - 500 and was not limited to 100 fields to be sure of the status of the samples . to the best of our knowledge , this method is the most sensitive method reported till date , having a detection limit of 300 - 400 bacilli per ml of sample . ( fig3 ). 4 . the sensitivity ( limit of detection ) can be further increased by using more than the prescribed 10 % of the processed specimen for smear microscopy . 5 . in case culture and pcr tests are not performed , & gt ; 10 % of the sample can be examined on multiple slides . this approach is expected to further improve the sensitivity of smear microscopy by usp method . 6 . the method is also compatible with mycobacteria other than m . tuberculosis . 7 . the method of smear microscopy is applicable on all mycobacteria including slow growing species like organisms belonging to the m . tuberculosis complex , m . avium , m . intracellulare , m . paratuberculosis m . xenopi , etc . rapid growing species like m . smegmatis , m . fortuitum , m . phlei , m . vaccae , m . gordonae etc . and non - cultivable species like m . leprae . 8 . the usp smear microscopy method is compatible with gram positive organism such as staphylococcus sp and is predicted to be applicable on other acid - fast bacilli ( e . g . cryptosporidium sp ., nocardia sp ., isospora belli and rhodococcus equi .) 9 . highly sensitive : the process effectively removes the background counterstaining material from the samples , enabling smearing of at least 10 % of the sample on the slide , which would have been impossible otherwise . owing to such efficient removal of the counterstaining junk , smearing as much as 10 % of the sample also does not produce a thick smear and the smear can be very easily heat - fixed and stained ( fig9 b ). 10 . tissue biopsy samples can be directly processed for smear microscopy bypassing the routine histologic procedures of fixing , paraffin - embedding and sectioning . 1 . the usp solution serves as a very effective decontamninant and is suitable for use in tropical countries with humid climate . 2 . the process does not involve the use of costly reagents like nalc . 3 . the usp solution is a buffered solution having neutral ph and does not involve the use of naoh . so neutralization of the sample is not necessary before culturing . sample neutralization is a prime requirement in methods involving decontamination with naoh ( such as cdc method ). improper neutralization often adversely affects culture results . 4 . the process is versatile and can be used for culturing m . tuberculosis from a wide range of pulmonary and extra - pulmonary samples in addition to sputum . 5 . the method is also compatible with culture of mott bacilli . 1 . this method being a multipurpose method , no separate method is needed for isolating dna from the samples processed for smear microscopy and culture . 2 . this method efficiently removes pcr inhibitors . inhibitor - free pcr - amplifiable dna can be reproducibly isolated from all types of pulmonary and extra - pulmonary samples . 3 . it does not involve the use of hazardous organic solvents like phenol or chloroform or expensive enzymes like lysozyme ; etc . 4 . the dna . isolation method would be compatible with nucleic acid amplification methods other than pcr such as ligase chain reaction , strand displacement assay , etc . 5 . a specific and sensitive pcr assay is described that enables sensitive and specific diagnosis of tuberculosis . 6 . compared to the irs method of dna isolation [ chakravorty and tyagi 2001 ; inhibitor removal solution ( irs ) is covered under - patent application entitled “ a rapid , efficient , single — tube extraction procedure for the isolation of pcr — amplifiable m . tuberculosis dna from clinical specimens ”, filed in patents office , new delhi . appln . no . 497 / deu2000 . ], the usp method is much simplified and involves lesser technology . firstly , the centrifugation can be performed in table - top centrifuges which can go up to a maximum speed of 4000 rpm and at room temperature , thereby avoiding the necessity of a high speed refrigerated centrifuge . secondly , the use of nalc as a mucolytic agent , is eliminated , thereby saving on cost . thirdly , the usp method is environment friendly as it does not involve the use of guanidinium isothiocyanate . fourthly , the usp method does not require the use of lysozyme enzyme . 7 . the dna isolation method is also compatible with nucleic acid amplification methods employed for the detection of other mycobacteria . 8 . since the usp processing method does not involve treatment with alkali , it is also compatible with rna isolation procedures from clinical specimens and rna amplification procedures such as transcription - mediated amplification . 1 . usp - treated mycobacteria are compatible for use with standard rna - isolation protocols . 2 . rna isolated thereby are suitable for further analysis by amplification techniques employing enzymes such as reverse transcriptase , taq dna polymerase etc . 3 . both mrna and rrna are successfully isolated from usp - treated m . tuberculosis . 1 . akhtar m , bretzel g , boulahbal f , dawson d , pattorini l , feldmann k , frieden t , havelková m . n de kantor i , kim s j , küchler r , iamothe f , laszlo a , casabona n m , mcdougall a c , miörner h . orefici g , paramasivan c n , pattyn s r , reniero a , rieder h l , ridderhof j , rüsch - gerdes s , siddiqi s h , spinaci s , urbanczik r , vincent v , weyer k ( 2000 ) technical guide : sputum examination for tuberculosis by direct microscopy in low income countries 5th edition , international union against tuberculosis and lung disease , 68 boulevard saint michel , 75006 paris , france . 2 . al - soud w a , radstrom p . 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