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Journal of Clinical Microbiology, June 2005, p. 2697-2702, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2697-2702.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Multipurpose Methodology for Detection of Mycobacteria in Pulmonary and Extrapulmonary Specimens by Smear Microscopy, Culture, and PCR{dagger}

Soumitesh Chakravorty{ddagger} and Jaya Sivaswami Tyagi*

Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

Received 12 February 2004/ Returned for modification 6 September 2004/ Accepted 13 February 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A novel, robust, reproducible, and multipurpose universal sample processing (USP) methodology for highly sensitive smear microscopy, culturing on solid and liquid media, and inhibition-free PCR which is suitable for the laboratory diagnosis of both pulmonary and extrapulmonary tuberculosis (TB) has been developed. This method exploits the chaotropic properties of guanidinium hydrochloride for sample processing and involves incubating the specimen with USP solution, concentrating bacilli by centrifugation, and using the processed specimen for smear microscopy, culture, and PCR. The detection limit for acid-fast bacilli in spiked sputum by smear microscopy is approximately 300 bacilli per ml of specimen. USP solution-treated specimens are fully compatible with culturing on solid and liquid media. High-quality, PCR-amplifiable mycobacterial DNA can be isolated from all types of clinical specimens processed with USP solution. The method has been extensively validated with both pulmonary and extrapulmonary specimens. Furthermore, the USP method is also compatible with smear microscopy, culture, and PCR of mycobacteria other than tubercle bacilli. In summary, the USP method provides smear microscopy, culture, and nucleic acid amplification technologies with a single sample-processing platform and, to the best of our knowledge, is the only method of its kind described to date. It is expected to be useful for the laboratory diagnosis of TB and other mycobacterial diseases by conventional and modern methods.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tuberculosis (TB) kills more people in India and Southeast Asia than any other infectious disease—more than human immunodeficiency virus, sexually transmitted diseases, malaria, and tropical diseases combined. In India, 1 person dies every minute and 500,000 people die per year from TB (37). The rapid diagnosis of TB is central to minimizing the risk of disease transmission, especially in the wake of the emergence of drug-resistant TB and its severe implications for human immunodeficiency virus-infected patients (37). Although culturing of the etiologic agent remains the accepted "gold standard" for diagnosing TB, direct smear microscopy is by far the most popular among all the methods currently employed worldwide for TB diagnosis. In view of the enormous utility of smear microscopy, numerous efforts have been made to improve its sensitivity (11, 13, 18, 27, 31, 36), with varying success rates. In recent times, however, maximum attention has been devoted to developing nucleic acid amplification (NAA) diagnostic technologies owing to their rapidity and sensitivity. Numerous gene targets (10, 29, 35) and several methods for isolating mycobacterial DNA from clinical specimens have been reported (2, 9, 19, 23, 26, 32). Several automated and semiautomated kits for mycobacterial culture and NAA are available and are being extensively evaluated (3, 5, 14, 15, 20, 30).

Despite the availability of a plethora of tests, there are certain drawbacks associated with the existing diagnostic methodologies. Direct smear microscopy lacks sensitivity and versatility in terms of application to extrapulmonary specimens. Methods other than those employing CB-18 (36) and N-acetyl L-cysteine (NALC)-NaOH (a method developed at the Centers for Disease Control [also referred to here as the CDC method]) (18) are suitable mainly for smear microscopy but not for culture. Automated and semiautomated culture systems are relatively rapid, but they involve sophisticated, expensive, or hazardous technologies, which is a deterrent to their widespread application in countries where the disease is endemic and resources are scarce, such as India. Lastly, there is no universal environmentally friendly method for isolating mycobacterial DNA from clinical specimens that guarantees the isolation of PCR-quality inhibitor-free mycobacterial DNA.

Against this background, the universal sample processing (USP) methodology has been developed, which includes a unique and simple sample processing technique that is compatible with the three most commonly used laboratory methods of TB diagnostics, namely, smear microscopy, culture, and PCR (J. S. Tyagi and S. Chakravorty, PCT publication no. WO 2005010186 dated 3 February 2005). It is applicable to all types of clinical material of pulmonary and extrapulmonary origins. This method uses nontoxic guanidinium hydrochloride as the principal component, in conjunction with a mucolytic agent and detergents. The method is also compatible with mycobacterial RNA isolation from clinical specimens, which will make it compatible with NAA diagnostic procedures employing RNA.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Specimen processing by USP methodology. USP solution consists of 4 to 6 M guanidinium hydrochloride (GuHCl), 50 mM Tris-Cl, pH 7.5, 25 mM EDTA, 0.5% Sarkosyl, and 0.1 to 0.2 M ß-mercaptoethanol (all chemicals were purchased from Sigma Aldrich, Bangalore, India).

(i) Sputum. USP solution (1.5 to 2 volumes) was added to each sputum sample, followed by vortexing for 30 to 40 seconds (optional) or shaking by hand for 1 to 2 min. The homogenate was incubated for 5 to 10 min at room temperature, after which 10 to 15 ml of sterile water was added and mixed. Bloodstained, highly purulent, and/or tenacious sputum was incubated for an additional 10 min. The sample was centrifuged at 5,000 to 6,000 x g at room temperature for 10 to 15 min, and the supernatant was discarded carefully. If the sediment size did not reduce appreciably (to 5 to 10% of the original sputum volume), the sample was washed once more with 2 to 5 ml of USP solution followed by a wash with 10 ml of sterile triple-distilled water. The sediment was used for smear microscopy, culture, and DNA/RNA isolation for PCR/reverse transcriptase PCR (RT-PCR).

(ii) Blood. An equal volume of USP solution was added to each blood sample (containing EDTA) and mixed well, and no further incubation was necessary. For partially clotted blood, samples were incubated at 37°C in USP solution for 15 to 30 min, depending on the extent of the clot present. Thereafter, the samples were processed in the same manner as that for sputum samples (addition of water, same centrifugation speed, etc.). If necessary, the sediment was washed once or twice with USP solution until a buff-colored sediment was obtained, followed by a wash with sterile triple-distilled water.

(iii) Other body fluids (except sputum). Pleural fluids were collected in EDTA vials to prevent the formation of coagulum. Samples were pelleted at a medium speed (5,000 to 6,000 x g), and the pellets were processed in the same manner as that for sputum samples (USP treatment, addition of water, same centrifugation speed, etc.). For very thick pus samples, 1 to 2 volumes of USP solution was added and mixed by vortexing, the samples were incubated for 15 to 20 min at 37°C, and some water was added to the samples before centrifugation and pelleting. In the case of milk, fat was removed with a sterile cotton swab after centrifugation at 25,000 x g. The sediment was washed once or twice with 1.5 to 2 volumes of USP solution, followed by a wash with sterile triple-distilled water. Cerebrospinal fluid specimens were pelleted at 25,000 x g and given a USP wash followed by a water wash.

(iv) Tissue biopsy (fresh or embedded in paraffin). Each tissue biopsy was incubated with USP solution for 15 to 20 min, minced as finely as possible (for fine-needle biopsies, mincing was not necessary), and subjected to disintegration in a mini-bead beater (Biospec Products) for 30 to 90 seconds in the presence of 0.5 to 1 volume of sterile 1-mm glass beads (in a total volume of 1 ml). The vial was briefly centrifuged at 200 to 400 x g and the homogenate (supernatant) was recentrifuged at 25,000 x g for 10 min. The recovered sediment was washed once again with USP solution, followed by a wash with sterile water. For paraffin-embedded tissue, deparaffinization with xylene was performed, as described previously (41), before USP processing.

USP smear microscopy, culture, and DNA isolation techniques. The USP sediment obtained after the water wash was resuspended well in 500 µl of sterile 0.05% Tween 80 (hereafter referred to as resuspension solution) and then transferred to a 1.5-ml polypropylene vial. For smear examination, 50 µl of the suspension was smeared onto a clean glass slide in a minimal area generally not exceeding 1 cm by 1 cm; for culture, 225 µl was inoculated onto LJ medium or MGIT and incubated at 37°C. For DNA isolation, 225 µl of the suspension was pelleted at 20,000 x g in a 1.5-ml microcentrifuge tube at room temperature. A solution made up of ~5 to 6 volumes of 10% Chelex-100 resin containing 0.03% Triton X-100 and 0.3% Tween 20 was added to the sediment. The contents were mixed well, heated at 90°C for 40 min, and centrifuged at room temperature at 20,000 x g for 5 min. If the sediment size was drastically reduced upon USP treatment or was very minute or if the sample had an initial high bacillary load, lysis was carried out by heating the suspension directly or after adding Triton X-100 (final concentration, 0.1%) at 90°C for 40 min. The supernatant was used directly for PCR. Triton X-100 was used as a simple and cheap alternative to the lysis reagent when the final pellet size was very minute (indicating an efficient removal of PCR inhibitors by USP processing and/or that the initial bacterial load in the specimen was very high) so that even a relatively less efficient lysis protocol would yield enough PCR-amplifiable mycobacterial DNA. All centrifugations were performed in fixed angle rotors for efficient recovery of the sediment.

Detection limit of USP smear microscopy. A logarithmic-phase Mycobacterium tuberculosis culture was serially diluted in duplicate sets to 106-fold in sterile Middlebrook 7H9 medium containing 0.05% Tween 80. Ten percent of each dilution from set 1 was inoculated onto an LJ slant to estimate the CFU, and the dilutions from set 2 were used to quantitatively spike 1-ml sputum aliquots from a patient suffering from chronic obstructive pulmonary disease. Prior to spiking, the sputum was homogenized by being vortexed in the presence of five or six sterile 4- to 5-mm glass beads and then allowed to stand for 5 to 10 min prior to collection of the homogenate in a separate tube. The sputum homogenate was confirmed to be negative for acid-fast bacilli (AFB) by smear microscopy of multiple slides prepared from a 5-ml portion processed by the USP method as well as by IS6110 PCR (10) analysis of the DNA isolated from the sample. The spiked samples were processed by the USP method, and 10% of each sample was smeared onto duplicate glass slides and examined for AFB by Ziehl-Neelsen (ZN) staining. The same experiment was also performed with a Mycobacterium smegmatis culture.

Effect of USP solution treatment on the viability and integrity of tubercle bacilli. For an evaluation of the effect of USP treatment on viability, an M. tuberculosis culture was serially diluted to 104-fold in duplicate in sterile Middlebrook 7H9 medium containing 0.05% Tween 80. Ten percent of each dilution from set 1 was inoculated onto an LJ slant, and the dilutions from set 2 were used to quantitatively spike 1-ml aliquots of homogenized sputum (previously determined to be AFB negative and culture negative as described above) from a patient suffering from chronic obstructive pulmonary disease. The spiked samples were processed by the USP method as described above, and 10% of the processed material was inoculated onto LJ slopes.

To monitor the integrity of tubercle bacilli, we serially diluted an M. tuberculosis culture to 106-fold in triplicate sets in sterile Middlebrook 7H9 medium containing 0.05% Tween 80. The bacteria were pelleted in two dilution sets by centrifugation. The sediments from set 1 were treated with 500 µl of USP solution for 30 min at room temperature, followed by centrifugation. DNAs were isolated from both set 1 and set 2 (untreated) sediments by boiling for 30 min at 100°C in the presence of 0.1% Triton X-100. DNAs were also isolated from the supernatants by the phenol-chloroform method as described previously (22) and were used in IS6110-based PCRs to assess bacterial lysis. Set 3 dilutions were plated on Middlebrook 7H10 agar to determine the numbers of CFU.

Effect of USP on growth rate of M. tuberculosis. An M. tuberculosis culture was divided into three sets. Set 1 was treated with 1.5 to 2 volumes of USP solution for 30 min at room temperature, followed by a wash with sterile water. Set 2 was treated with an equal volume of 1.0% NALC-4% NaOH for 15 min, followed by neutralization with 68 mM phosphate buffer, pH 6.8 (24). All three sets, i.e., set 1, set 2, and set 3 (untreated), were inoculated onto LJ medium and into MGIT tubes with the PANTA reagent and an oleic acid-albumin-dextrose-catalase supplement (Becton Dickinson) and then monitored for positive signals by the BD BACTEC MGIT 960 system.

PCR. IS6110-specific PCRs (10) were performed in 40-µl reaction mixtures containing a 0.5 µM concentration (each) of the primers T4 and T5, 1x PCR buffer [PCR buffer with (NH4)2SO4; MBI Fermentas, Vilnius, Lithuania], 1.5 mM MgCl2, a 0.2 mM concentration of each deoxynucleoside triphosphate, 1 U of Taq DNA polymerase (GeneTaq; MBI Fermentas, Vilnius, Lithuania), and 10 µl of specimen DNA. The thermal cycling parameters were 10 min at 94°C, 45 cycles of 1 min at 94°C and 1 min 30 seconds at 60°C, and a final extension of 7 min at 72°C. PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis. PCR inhibition was assessed by adding 20 to 30 ng of pure M. tuberculosis DNA into reactions containing specimen DNA.

Mycobacterial RNA isolation from sputum and RT-PCR analysis. RNAs were isolated from USP-treated sediments by use of an RNeasy mini kit (QIAGEN GmbH, Hilden, Germany). M. tuberculosis RNAs were reverse transcribed with the Stratascript reverse transcriptase (RT) enzyme (Stratagene Inc., La Jolla, Calif.), and the cDNAs generated were subjected to PCR amplification with M. tuberculosis 23S rRNA- and Rv3134c mRNA-specific primers, as described previously (16, 38).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A flowchart of the USP processing and diagnostic methodology is shown in Fig. 1.



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  		FIG. 1. Outline of USP processing protocol.

 
The principal components in USP solution act in a synergistic manner, resulting in optimal specimen processing. The contributions of individual components of the USP solution were assessed with respect to all three diagnostic modalities. First, the USP method was standardized and optimized by using sputum samples with differing physical characteristics (mucoid, purulent, watery, induced, tenacious, bloodstained, etc.) and bacillary loads (scanty to a score of 3+ by the direct method of smear microscopy). There was no reduction in the sediment size when sputa were processed with USP solution lacking the three principal components, GuHCl, Sarkosyl, and ß-mercaptoethanol, in various combinations. This was indicative of inefficient removal of extraneous cellular debris and background material from the samples. Excessively thick smears with heavily counterstaining backgrounds resulted when 10% of the final processed sediments was smeared onto slides, which either were very difficult to read or scaled off during ZN staining due to improper fixation of the smear. Furthermore, decolorization of the medium and contamination were noted within 2 days of inoculation on LJ medium, and PCRs showed inhibition. Contamination was not detected when the specimens were processed with complete USP solution or variations thereof containing GuHCl but lacking ß-mercaptoethanol or Sarkosyl, and all of the cultures were positive for M. tuberculosis at the end of 3 weeks. Thus, the absence of any one of the principal components resulted in suboptimal processing of the sputum specimens. Among the variations in the USP solution, the complete formulation always fared the best in terms of the removal of (i) cellular debris, thereby producing the cleanest sediments, and (ii) PCR inhibitors, thereby leading to reproducible and efficient DNA amplification. The USP method was equally efficient at processing sputa with different physical characteristics, such as bloodstained, tenacious, purulent, or mucoid sputa (not shown). Thus, USP-processed bacillary sediments were clean and could be directly used for smear microscopy, culture, and DNA isolation for PCR. (7; PCT publication WO 2005010186).

USP solution has a negligible effect on the integrity of M. tuberculosis. The integrity of USP-treated M. tuberculosis (serial dilutions ranging from ~107 to 50 bacterial equivalents) was monitored by subjecting supernatant fractions of USP-treated bacilli to DNA isolation and PCR. After 30 min of exposure to USP solution, equivalent intensities of PCR products were noted for DNAs isolated from USP-treated and untreated bacterial sediments until a dilution of 10–5. Furthermore, amplifiable DNAs were not isolated from the supernatants beyond the 10–7 dilution range, indicating that a significant loss of bacterial DNA due to lysis by the USP solution did not occur. However, the DNA from the 10–6 dilution of USP-treated bacilli resulted in an amplification product with a lower intensity than that from untreated bacilli. This dilution contained 50 to 60 CFU/ml and a visible amplicon was obtained from 10 to 12 bacillary equivalents (the amount of DNA added to the PCR), even in the case of USP-treated bacilli (not shown).

USP smear microscopy can detect specimens containing as few as 250 to 300 AFB/ml. USP-treated bacteria, either from cultures or from the sputum milieu, retained their acid-fast property during ZN staining. In several experiments wherein M. tuberculosis-spiked sputa were processed by the USP method, ZN staining could reproducibly detect specimens containing as few as 250 to 300 AFB per ml (when at least 400 to 500 fields were read). This was ascribed to the concentration of the bacilli and the efficient removal of the counterstaining background, which enabled smearing of at least 10% of the processed material onto the slide. This limit of detection was reproducible for sputum spiked with M. smegmatis as well. In a representative experiment with M. tuberculosis-spiked sputum, the slides were reproducibly read as having a score of 2+/3+ down to a CFU of 104 AFB/ml. At CFU of 2 x 103 to 3 x 103 AFB/ml, the slides were reproducibly detected to have a score of 1+, and at CFU of 5 x 102 to 3 x 102 AFB/ml, the slides mainly belonged to the scanty category.

USP treatment is compatible with culture of tubercle bacilli on solid and in liquid media. After 8 weeks, the viable count of tubercle bacilli in USP-treated spiked sputum was 400 CFU/ml (at the 10–4 dilution), in contrast to 720 CFU/ml for the corresponding dilution of untreated cells. Thus, ~55% of the bacteria survived the treatment with USP. In a second independent experiment, ~38 to 44% of the bacteria survived a similar treatment. Treatments with decontaminating reagents such as NaOH are known to reduce the growth rate of M. tuberculosis from clinical material (20). A similar observation was also made with USP-treated bacteria; after USP treatment, colonies appeared on solid medium (Middlebrook 7H10 or LJ) after a lag time of ~7 to 10 days compared to untreated bacteria. In a parallel check, NALC-NaOH-treated bacteria grew somewhat faster (visible colonies were obtained 4 to 5 days earlier).

However, appreciable differences were not observed between the USP and NALC-NaOH treatments when in vitro-cultured bacteria were grown in the MGIT liquid culture system. In two independent experiments, untreated logarithmic-phase tubercle bacilli were positive for growth in 2 days, whereas both USP- and NALC-NaOH-treated cells were positive for growth in 5 days. This suggests that both the USP and CDC methods slow down the growth of M. tuberculosis in MGIT liquid medium to equivalent extents. The USP treatment was extended to include AFB-positive sputa and yielded positive cultures in the MGIT system, demonstrating its compatibility with culture in solid and liquid culture systems.

The USP methodology enables isolation from sputum specimens of high-quality, inhibitor-free mycobacterial nucleic acids which are compatible with amplification technologies. The utility of the USP methodology for isolating inhibitor-free M. tuberculosis DNA was established with clinical specimens received at the All India Institute of Medical Sciences (AIIMS) laboratory for routine diagnosis and also for validation purposes (6a; our unpublished data). PCR inhibition was routinely assessed by adding 20 to 30 ng of pure M. tuberculosis DNA to reaction mixtures containing specimen DNA; no inhibition was noted, which established the efficacy of the USP treatment for removing PCR inhibitors. In addition to DNA, rRNA and mRNA could also be amplified via RT-PCR from USP-treated sputum. Thus, the USP method of processing sputum specimens is also compatible with mycobacterial RNA isolation.

USP treatment of extrapulmonary specimens. The USP methodology was also standardized and optimized for use with various types of extrapulmonary specimens. These included transtracheal aspirates, bronchoalveolar lavage fluid, laryngeal swabs, nasopharyngeal swabs, body fluids such as pleural fluid, pericardial fluid, joint aspirates, gastric aspirates, peritoneal fluid, cerebrospinal fluid, urine, pus, endometrial aspirates, and synovial fluid, body tissues such as blood and bone marrow biopsies, solid organs such as lymph nodes, bones, and skin, and bovine samples, including lymph glands, milk, and blood. The method was evaluated on ~500 extrapulmonary specimens, including some sent to the AIIMS laboratory for routine TB diagnosis. USP-processed samples did not show PCR inhibition, and smear microscopy revealed AFB against minimally counterstaining backgrounds in cases of positive specimens. Moreover, the PCR and smear microscopy results showed a high degree of concordance with the working diagnosis and therapeutic response of the patients established at the hospitals from which the specimens originated (our unpublished data).

Thereafter, the standardized USP methodology was further validated in different clinical settings with a panel of 571 sputa (6a) and with 100 extrapulmonary specimens comprising pleural effusion, lymph node, and pleural tissue biopsies, yielding excellent results (S. Chakravorty, M. K. Sen, and J. S. Tyagi, submitted for publication).

USP treatment of MOTT bacilli. Several mycobacteria other than tubercle (MOTT) bacillus species, including M. avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. phlei, M. scrofulaceum, M. smegmatis, and M. vaccae, and other organisms belonging to the M. tuberculosis complex, including M. africanum, M. bovis, M. bovis BCG, and M. microti, were subjected to USP treatment as described above (see Materials and Methods). USP-processed pellets were subjected to smear microscopy, culture on LJ medium, and DNA isolation as described above. USP treatment did not alter the AFB properties or integrity of any of the mycobacteria examined, with the exception of M. smegmatis, which lost its viability after USP treatment. High-quality PCR-amplifiable DNAs were isolated from all MOTT bacilli. This was evident from the successful amplification of the gene encoding the 65-kDa protein by the use of previously described primers (35). In summary, USP treatment is compatible with MOTT bacilli and can be extended to processing clinical specimens containing these species. (7; PCT publication WO 2005010186).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here a rapid, reproducible, robust, inexpensive, and easy universal sample processing and diagnostic methodology applicable to any type of clinical specimen for the laboratory diagnosis of TB. It serves the purposes of (i) providing highly sensitive smear microscopy for AFB detection, (ii) preparing samples for culture on solid or in liquid media, and (iii) isolating inhibitor-free DNA/RNA templates for NAA tests. The methodology has been extensively evaluated on pulmonary and extrapulmonary clinical specimens (6a; Chakravorty et al., submitted). It comprises optimized sample processing incorporating a novel use for the chaotropic agent GuHCl. The unique properties of the mycobacterial cell wall render these organisms selectively resistant to the action of GuHCl, while cells of other bacteria and eukaryotic cells are disrupted upon exposure to it. The efficacy of USP solution on specimens with widely ranging physicochemical properties is attributed to the synergistic effects of its individual constituents, which perform decontaminating, protein-denaturing, membrane-disrupting, lipid-solubilizing, mucolytic, and PCR inhibitor and junk removal functions in a buffered environment.

One of the primary reasons for the insensitivity of direct smear microscopy (28) is the minuscule amount of specimen smeared on the slide (37). To the best of our knowledge, the present study reports the most sensitive smear microscopy method described to date, which is the first one to increase the volume of sample smeared on the slide without scaling off (up to 50 to 60% of the processed material [unpublished observations]). Removing cellular debris and counterstaining material without altering the staining properties of AFB minimized technical errors and reduced the time spent reading each slide. Specimens containing ~300 to 400 bacilli per ml were reproducibly detected as positive by USP smear microscopy, which is at least 12- to 16-fold more sensitive than the published limit of detection by smear microscopy (27). The practical benefits of USP smear microscopy would be especially relevant in laboratories routinely handling large sample loads. Per specimen, the cost of sputum microscopy was estimated to vary between U.S. $0.54 and 1.97 per slide (40) versus <$0.50 for USP smear microscopy and culture. Therefore, the USP method may have widespread utility in national TB control programs of economically vulnerable nations, whose first component is case detection by smear microscopy.

USP also confers advantages for culturing mycobacteria from clinical specimens. Since the solution is at a neutral pH, the cumbersome step of neutralizing decontaminated specimens before culturing is avoided. USP treatment rendered at least 45 to 66% of treated mycobacteria nonviable, which was not strikingly different from the amount noted following decontamination with 2 to 3% NaOH (21, 42). However, the USP solution containing 4 M GuHCl (in place of 5 to 6 M GuHCl) was observed to be less harsh to mycobacteria (unpublished observations); thus, when culturing M. tuberculosis is of prime importance, the use of USP solution containing 4 M GuHCl is likely to increase the positivity rate.

Numerous in-house methods for isolating PCR-amplifiable M. tuberculosis DNA have been described (2, 9, 8, 17, 23, 26, 32). Despite the use of various approaches (1, 4, 12, 39), most methods do not consistently remove PCR inhibitors from clinical material (12, 17, 33, 34, 43). Even commercial PCR kits like the Amplicor M. tuberculosis kit are vulnerable to PCR inhibition (4, 30). The USP method described in the present study enables the isolation of inhibitor-free high-quality mycobacterial DNA (as well as RNA) from a variety of clinical materials in an environmentally friendly, simple, and inexpensive manner. The selective washing away of inhibitors before the lytic treatment to release the DNA from tubercle bacilli relinquishes the need for an additional DNA purification step.

The utility and reliability of NAA tests are widely debated, especially for smear-negative samples (6). Double-blind studies have raised doubts about the reliability of "in-house" PCR as a routine diagnostic procedure (24, 25), relegating PCR to the status of an adjunct test. A rigorous evaluation of PCR techniques necessitates a comparison of their performances with that of smear microscopy, culture, or both. This oftentimes leads to erroneous analyses of NAA tests due to differences arising out of variations in sample processing methods and nonhomogeneous portioning of specimens for the individual tests. The USP technology has enabled a reliable validation of NAA-based tests in clinical settings as the currently accepted gold standard, and PCR-based tests can be performed on an entire sample by using one processing methodology, precluding the need for a dedicated method to isolate DNA for PCR (6a; Chakravorty et al., submitted).

In summary, we have described a methodology that can be reliably applied to all types of clinical specimens for diagnosing TB in laboratories with diverse infrastructure capabilities. Initial studies from our laboratory have also indicated that the methodology may be suitable for smear microscopy and culture of gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae (not shown). Until now, methods developed to enhance the performance of TB diagnostics have focused on improving one or another existing conventional methods, whereas the USP method has attempted to provide a simple, rapid, and extremely reproducible multipurpose technique compatible with all common diagnostic modalities at an affordable cost. Because of the features described here, the USP technology is highly suitable for the diagnosis of TB and other mycobacterial diseases.


   ACKNOWLEDGMENTS
 
We thank V. D. Ramanathan, Tuberculosis Research Centre, Chennai, India, for providing paraffin-embedded guinea pig skin tissue infected with heat-killed M. tuberculosis and Sarman Singh, Department of Laboratory Medicine, AIIMS, for the use of the BD BACTEC MGIT 960 system. We thank Sanjay Kumar for his technical assistance during the study. J.S.T. is thankful to P. K. Dave for encouragement and support.

S.C. is thankful to I.C.M.R. for a senior research fellowship. Financial support to J.S.T. from AIIMS is acknowledged.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India. Phone: 91-11-26588491. Fax: 91-11-26588663. E-mail: jstyagi{at}aiims.ac.in. Back

{dagger} This work is dedicated to the memory of T. A. Venkitasubramanian, who contributed greatly to the study of mycobacterial metabolism. Back

{ddagger} Present address: Division of Infectious Diseases, Department of Medicine, and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Clinical Microbiology, June 2005, p. 2697-2702, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2697-2702.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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