Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues: comparison of four amplification assays

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the mtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 microg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.     

Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens

The rapid identification of mycobacterial DNA in clinical samples by PCR can be useful in the diagnosis of tuberculous infections, but several large studies have found that the sensitivity of this approach is not better than that of culture. In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients with paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other potential inhibitors of the amplification reaction (sequence capture-PCR). By using this approach to capture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat region), it was possible to detect as little as one genome of mycobacterial DNA in samples containing up to 750 micrograms of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification. Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleurisy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negative samples. In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the three culture-positive samples were positive by PCR. The sensitivity of detection of the direct repeat sequence in these samples by sequence capture-PCR was similar to that of IS6110 and, in addition, permitted immediate typing of the strains from some patients. We conclude that sequence capture-PCR improves the sensitivity of detection of mycobacterial DNA in paucibacillary samples. This approach should be useful in detecting rare target sequences from organisms implicated in other pathologic processes.     

Acute effects of the oral administration of midodrine, an alpha-adrenergic agonist, on renal hemodynamics and renal function in cirrhotic patients with ascites

The successful use of DNA amplification for the detection of tuberculous mycobacteria crucially depends on the choice of the target sequence, which ideally should be present in all tuberculous mycobacteria and absent from all other bacteria. In the present study we developed a PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system named SenX3-RegX3. The senX3-regX3 IR is composed of a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). In a survey of 116 Mycobacterium tuberculosis strains characterized by different IS6110 restriction fragment length polymorphisms, 2 Mycobacterium africanum strains, 3 Mycobacterium bovis strains (including 2 BCG strains), and 1 Mycobacterium microti strain, a specific PCR fragment was amplified in all cases. This collection included M. tuberculosis strains that lack IS6110 or mtp40, two target sequences that have previously been used for the detection of M. tuberculosis. No PCR fragment was amplified when DNA from other organisms was used, giving a sensitivity of 100% and a specificity of 100% in the confidence limit of this study. The numbers of MIRUs were found to vary among strains, resulting in six different groups of strains on the basis of the size of the amplified PCR fragment. However, the vast majority of the strains (approximately 90%) fell within the same group, containing two 77-bp MIRUs followed by one 53-bp MIRU.     

PCR-based rapid detection of Mycobacterium tuberculosis in blood from immunocompetent patients with pulmonary tuberculosis

A PCR test based on insertion sequence IS1081 was developed to detect Mycobacterium tuberculosis complex organisms in the peripheral blood. The method was applied to blood samples from immunocompetent individuals with localized pulmonary tuberculosis. Seven of 16 (43.75%) blood samples were found to be positive for the circulating DNA copies of M. tuberculosis complex.     

Molecular fingerprinting of mycobacterium tuberculosis isolates obtained in havana, cuba, by IS6110 restriction fragment length polymorphism analysis and by the double-repetitive-element PCR method

Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosis strains in laboratories not equipped to perform RFLP analysis.     

Neuroendocrine small cell uterine cervix cancer in pregnancy: long-term survival following combined therapy

The presence of enterobacterial repetitive intergenic consensus (ERIC) sequences was demonstrated for the first time in the genome of Mycobacterium tuberculosis; these sequences have been found in transcribed regions of the chromosomes of gram-negative bacteria. In this study genetic diversity among clinical isolates of M. tuberculosis was determined by PCR with ERIC primers (ERIC-PCR). The study isolates comprised 71 clinical isolates collected from Sardinia, Italy. ERIC-PCR was able to identify 59 distinct profiles. The results obtained were compared with IS6110 and PCR-GTG fingerprinting. We found that the level of differentiation obtained by ERIC-PCR is greater than that obtained by IS6110 fingerprinting and comparable to that obtained by PCR-GTG. This method of fingerprinting is rapid and sensitive and can be applied to the study of the epidemiology of M. tuberculosis infections, especially when IS6110 fingerprinting is not of any help.     

Stability of Mycobacterium tuberculosis IS6110 restriction fragment length polymorphism patterns and spoligotypes determined by analyzing serial isolates from patients with drug-resistant tuberculosis

The stability of Mycobacterium tuberculosis IS6110 fingerprint patterns and spoligotypes has been assessed by analyzing serial isolates from patients with drug-resistant tuberculosis. Altogether, 165 M. tuberculosis isolates obtained from 56 patients have been analyzed. The time spans between the first and the last or a changed isolate from one patient ranged from 1 to 772 days. Among the 56 patients, 5 (9%) were infected with isolates with changes in their IS6110 fingerprint patterns. According to the total number of strains analyzed, 5% of the subsequent isolates showed variations in their IS6110 restriction fragment length polymorphism patterns compared to the pattern of the first isolates. Up to 10 isolates from one patient sampled at time intervals of up to 772 days with no changes in their IS6110 patterns have been analyzed. A statistically significant correlation could be found between changes in insertion sequence (IS) patterns and the increased time intervals over which the isolates were obtained, whereas changes in IS patterns are not correlated to changes in the drug resistance of the isolates. In contrast to the observed variations in IS6110 fingerprint patterns, no changes in the spoligotypes of the isolates analyzed could be found. In conclusion, our results confirm that the IS6110 fingerprint patterns of M. tuberculosis isolates have high degrees of stability. Compared to IS6110, the direct repeat (DR) region, which is the basis for spoligotyping, has a lower rate of change. Partial deletions, e.g., deletions induced by homologous recombination between the repetitive DR elements, could not be detected in this study.     

Characterization of IS1547, a new member of the IS900 family in the Mycobacterium tuberculosis complex, and its association with IS6110

Unlike classically defined insertion sequence (IS) elements, which are delimited by their inverted terminal repeats, some IS elements do not have inverted terminal repeats. Among this group of atypical IS elements, IS116, IS900, IS901, and IS1110 have been proposed as members of the IS900 family of elements, not only because they do not have inverted terminal repeats but also because they share other features such as homologous transposases and particular insertion sites. In this study, we report a newly identified IS sequence, IS1547, which was first identified in a clinical isolate of Mycobacterium tuberculosis. Its structure, insertion site, and putative transposase all conform with the conventions of the IS900 family, suggesting that it is a new member of this family. IS1547 was detected only in isolates of the M. tuberculosis complex, where it had highly polymorphic restriction fragment length polymorphism patterns, suggesting that it may be a useful genetic marker for identifying isolates of the M. tuberculosis complex and for distinguishing different strains of M. tuberculosis. ipl is a preferential locus for IS6110 insertion where there are eight known different insertion sites for IS6110. Surprisingly, the DNA sequence of ipl is now known to be a part of IS1547, meaning that IS1547 is a preferential site for IS6110 insertion.     

Comparison of two different PCR detection methods. Application to the diagnosis of pulmonary tuberculosis

The objectives are to assess the influence of the detection of the amplified DNA fragment on the sensitivity and specificity of the polymerase chain reaction (PCR). One hundred seventy-five sputum samples from 123 patients were processed. Sixty samples were taken from 60 subjects without tuberculosis, and the rest were taken from subjects with tuberculosis confirmed by culture. A fragment of the IS6110 sequence of Mycobacterium tuberculosis, which was detected using two different methods, was amplified. The detection methods used were a digoxigenin-labeled specific probe and chemiluminescent development and reamplification (nested PCR) combined with agarose gel electrophoresis. Sensitivity with probe detection was 75.65% and specificity 100%. Using the nested PCR technique, sensitivity rose to 93.04%, but specificity decreased to 96.6%. PCR is a quick and adequate way to diagnose pulmonary tuberculosis in cases where staining is negative yet there is a clinical suspicion of tuberculosis, even though a standardization process and large scale evaluation are still needed to determine its true usefulness.     

Comparison of the amplified Mycobacterium tuberculosis (MTB) direct test, Amplicor MTB PCR, and IS6110-PCR for detection of MTB in respiratory specimens

Several nucleic acid amplification techniques (NAAT) have been developed for rapid and direct detection of Mycobacterium tuberculosis (MTB) from clinical specimens. This study compared the performances of the Gen-Probe Amplified MTB Direct Test (AMDT), Roche Amplicor MTB PCR test, and an IS6110-PCR assay with acid-fast smear and culture in the detection of MTB from 428 respiratory specimens from 259 patients. Patients' charts were reviewed for clinical correlation. Of 98 specimens that were clinically positive for MTB, acid-fast smear was positive in 50% of cases, culture in 93%, IS6110-PCR in 83%, AMDT in 84%, and Amplicor MTB PCR in 80%. Of 337 specimens that were negative for MTB, 117 (35%) were positive for nontuberculous mycobacteria. Specificities were as follows: smear, 89%; culture, 100%; IS6110-PCR, 99%; AMDT, 98%; and Amplicor MTB PCR, 96%. The accuracies of the tests were 80%, 98%, 96%, and 92%, respectively. MTB culture-positive specimens that were smear-negative were detected by AMDT and IS6110-PCR in 77% of cases and by Amplicor MTB PCR in 70%. NAAT was less sensitive than was culture for detection of MTB, but all these techniques had acceptable accuracy and were completed within hours. NAAT may be useful for rapid screening of respiratory specimens to distinguish MTB from nontuberculous mycobacteria infection in order to isolate patients.     

Measuring lateral skin temperature profile of premature infants in incubators with thermography

Tuberculosis is still one of the most widespread infection known to mankind. Although lung is the predominant site of disease, a sizeable population in Pakistan gets intestinal disease. Clinical presentation, radiologic and endoscopic examination provide clues to the diagnosis. However, a definitive diagnosis requires biopsy material with granulomas and/or caseation complemented by acid fast staining and culture. There are many occasions when biopsy material is scanty and even in some intestinal resection cases histologic evaluation fails to confirm or rule out tuberculosis. Therefore, an investigation was conducted to assess the efficacy of PCR in the detection of mycobacterial DNA in paraffin embedded intestinal tissue. In this study 12 histologically confirmed cases of intestinal tuberculosis and 2 cases with non specific inflammation but clinically suspected for abdominal tuberculosis were selected. One case of rectal polyp was included to serve as a negative control. M. tuberculosis DNA was amplified in 8 out of 12 histologically confirmed cases and in 2 cases diagnosed with non specific inflammation. Amplified products were obtained in 6 out of 10 PCR positive specimens with IS6110 region specific primers while 4 samples were negative, suggesting the absence of insertion sequence 6110 in these strains. However, amplification was obtained in these negative specimens with a second primer pair confirming them as M. tuberculosis complex species. On the basis of this study we conclude that; (1) Processed and paraffin embedded tissue material is suitable for PCR analysis, (2) PCR assay can be used to complement the diagnosis of intestinal tuberculosis especially in situations where a definite conclusion can not be drawn by conventional methods, (3) M. tuberculosis species lacking insertion sequence 6110 element are present in our population. Therefore, several primer pair sets should be included when applying PCR for the detection of mycobacterial DNA.     

Infective endocarditis presenting as polyarthritis

IS6110 is commonly used as the basis for molecular epidemiologic and diagnostic studies of Mycobacterium tuberculosis. However, strains that do not contain IS6110 have been reported. If common, such strains would pose a limitation for molecular studies of M. tuberculosis. Analysis of a population-based sample from San Francisco of 1569 specimens submitted for fingerprinting demonstrated that the proportion of strains that lack IS6110 is less than 1%. While this low percentage permits IS6110 fingerprinting in San Francisco, it may be problematic in other settings.     

Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.     

Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.     

Spoligotyping followed by double-repetitive-element PCR as rapid alternative to IS6110 fingerprinting for epidemiological studies of tuberculosis

A total of 129 clinical isolates of Mycobacterium tuberculosis representing 91 patients were typed by a combination of direct-repeat (DR)-based spoligotyping and an inter-IS6110-PGRS (polymorphic GC-rich region)-PCR, also designated double-repetitive-element PCR (DRE-PCR). During the first phase of this investigation, 72 clinical strains representing 52 patients were initially typed by IS6110-restriction fragment length polymorphism (RFLP) and DR-RFLP, followed by spoligotyping and DRE-PCR. In the second phase of this investigation, the discriminating ability of spoligotyping plus DRE-PCR was studied for 57 isolates from 39 patients who were suspected to be epidemiologically linked, and the typing results were later confirmed by IS6110-RFLP and DR-RFLP analyses. The molecular clustering of the isolates remained identical irrespective of the methods used. These results show that the association of two PCR-based fingerprinting techniques for molecular epidemiology of tuberculosis has a discriminating ability similar to the IS6110-RFLP reference method.     

Comparison of C18-carboxypropylbetaine and glass bead DNA extraction methods for detection of Mycobacterium bovis in bovine milk samples and analysis of samples by PCR

The purpose of this prospective study was to compare two different milk preparation methods to assay for the presence of Mycobacterium bovis by PCR. Detection by a C18-carboxypropylbetaine (CB-18)-based sample processing method was compared to extraction of DNA from milk with glass beads. Samples from 17 skin test-positive cattle were analyzed. Following CB-18 processing and glass bead extraction, the sensitivity of IS6110-based PCR was 94.1 and 58.8%, respectively (P < 0.025). Because CB-18 processing will permit the proficient use of PCR for diagnosis and surveillance of bovine tuberculosis, it will contribute to the more efficient detection and control of tuberculosis.     

PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells

Oligonucleotides derived from IS6110, an insertion sequence from Mycobacterium tuberculosis, have been covalently immobilized on polystyrene Covalink NH microwells to develop a sandwich and a competitive non-radioactive hybridization assay for the quantitative determination of the DNA fragments obtained by polymerase chain reaction (PCR). Using the appropriate standard DNA, the method can be employed for the quantitative analysis of PCR fragments. The sandwich assay can detect as little as 3 fmol of target DNA per well and the standard curve may be used with quantities ranging from 3 to 300 fmol per well. The competitive hybridization assay is less sensitive since it is quantitative between 100 and 8000 fmol per well. We show here that both kinds of assays can be used to identify M. tuberculosis strains isolated from clinical samples. The non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells involve few and simple operations, and are thus suitable for routine diagnosis. Moreover, when stored at 5 degrees C, precoated strips can still be used for hybridization up to at least 10 months.     

Restriction fragment length polymorphism and spacer oligonucleotide typing: a comparative analysis of fingerprinting strategies for Mycobacterium bovis

The combination of conventional investigation and DNA fingerprinting is yielding important insights into the epidemiology of Mycobacterium bovis infections. Various genetic markers used in restriction fragment length polymorphism (RFLP) have recently been exploited for fingerprinting of M. bovis isolates. The newly developed spacer oligonucleotide typing aimed to investigate the polymorphism of M. tuberculosis in the DR locus, has also been applied to the molecular typing of M. bovis isolates. This work compared the performance of the insertion sequence (IS) IS6110, IS1081 and the genetic elements polymorphic G + C-rich repeat (PGRS) and direct repeat (DR) used in RFLP analysis with spoligotyping using a group of 128 Spanish M. bovis isolates. In this study, the most sensitive technique for identifying polymorphism in M. bovis was PGRS-RFLP, closely followed by IS6110-RFLP. We propose several schemes for fingerprinting of these isolates, however, the clear geographical variations found by different authors makes the study of each local situation indispensable. An international consensus in the methods used would be desirable for efficient interlaboratory comparison of strains.     

Comparison of PCR, culture, and histopathology for diagnosis of tuberculous pericarditis

Nucleic acid amplification techniques for the diagnosis of tuberculosis (TB) are rapidly being developed. Scant work, however, has focused on pericardial TB. Using cryopreserved specimens from a prior study of pericarditis, we compared PCR to culture and histopathology for the diagnosis of tuberculous pericarditis in 36 specimens of pericardial fluid and 19 specimens of pericardial tissue from 20 patients. Fluid and tissue were cultured on Lowenstein-Jensen and Middlebrook solid media and in BACTEC radiometric broth. Tissue specimens were stained with hematoxylin-eosin, Ziehl-Neelsen, auramine O, and Kinyoun stains and were examined for granuloma formation and acid-fast bacilli. PCR was performed with both fluid and tissue with IS6110-based primers specific for the Mycobacterium tuberculosis complex by published methods. Sixteen of the 20 patients had tuberculous pericarditis and 4 patients had other diagnoses. TB was correctly diagnosed by culture in 15 (93%) patients, by PCR in 13 (81%) patients, and by histology in 13 of 15 (87%) patients. PCR gave one false-positive result for a patient with Staphylococcus aureus pericarditis. Considering the individual specimens as the unit of analysis, M. tuberculosis was identified by culture in 30 of 43 specimens (70%) from patients with tuberculous pericarditis and by PCR in 14 of 28 specimens (50%) from patients with tuberculous pericarditis (P > 0.15). The sensitivity of PCR was higher with tissue specimens (12 of 15; 80%) than with fluid specimens (2 of 13; 15%; P = 0.002). In conclusion, the overall accuracy of PCR approached the results of conventional methods, although PCR was much faster. Therefore, PCR merits further development in this regard. The sensitivity of PCR with pericardial fluid was poor, and false-positive results with PCR remain a concern.     

Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR

A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population.