Antituberculosis IgG Antibodies as a Marker of Active Mycobacterium tuberculosis Disease

Anti-Mycobacterium tuberculosis IgG antibodies may aid in the diagnosis of active M. tuberculosis disease. We studied whether anti-M. tuberculosis IgG antibodies are elevated in active M. tuberculosis disease and assessed factors contributing to false-positive and -negative results. A retrospective study of 2,150 individuals tested by the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay was conducted at the University of Utah, ARUP Laboratories, November 2008 to December 2010. All samples were tested with the InBios Active TbDetect antituberculosis (anti-TB) IgG antibody assay. Of 1,044 patients with a positive QFT-GIT, 59 (5.7%) were positive for M. tuberculosis antibodies. Fourteen of 1,106 (1.3%) with a negative or indeterminate QFT-GIT were positive for M. tuberculosis antibodies. M. tuberculosis antibody tests were positive in 61.5% with confirmed active M. tuberculosis disease and other mycobacterial infections. Over half of the false-negative M. tuberculosis antibody tests occurred in patients ≥90 years of age. False positives were seen in 12.9% of autoimmune patients. The odds ratio of being positive by the QFT-GIT and the InBios TB IgG assay increased with confirmed M. tuberculosis disease or highly suspected M. tuberculosis disease and was 86.7 (95% confidence interval [CI], 34.4 to 218.5) in these two groups compared to patients negative by both tests. Although anti-M. tuberculosis antibodies can be detected in patients with active M. tuberculosis disease, caution should be used with patients where immunoglobulin levels may be decreased or patients with autoantibodies.     

Comparison of Roche Cobas Amplicor Mycobacterium tuberculosis Assay with In-House PCR and Culture for Detection of M. tuberculosis

The new Roche Cobas Amplicor Mycobacterium tuberculosis assay, which is a semiautomated version of the manually performed Roche Amplicor M. tuberculosis test, was compared to culture and an IS6110-based in-house PCR protocol. A total of 1,681 specimens from 833 patients, including specimen types other than sputum, were tested in parallel by both the in-house PCR and the Cobas Amplicor M. tuberculosis assay. After we resolved discrepant PCR results, the sensitivity, specificity, and positive and negative predictive values for the Cobas Amplicor M. tuberculosis assay were 66.33, 99.71, 94.36, and 97.66%, respectively. The corresponding values for the in-house PCR were 91.08, 99.85, 97.87, and 99.37%, respectively. For culture- and smear-positive specimens, the sensitivity of the Cobas Amplicor M. tuberculosis test was 96.42% (in-house PCR, 100%). If only smear-negative sputum specimens were considered, the Cobas Amplicor M. tuberculosis assay exhibited a sensitivity of 45.45% (in-house PCR, 63.63%) relative to that of culture. With a modified protocol for DNA extraction (washing of samples plus ultrasonication), both PCR methods performed better with gastric aspirates than with sputum samples (sensitivity of the Cobas Amplicor M. tuberculosis assay with smear-negative gastric aspirates, 70.00%; sensitivity of in-house PCR, 90.00%). With dithiothreitol being used for liquefaction of specimens in this study, the Cobas Amplicor M. tuberculosis assay exhibited an inhibition rate of 9.16%. In our view, the new Cobas Amplicor M. tuberculosis test (i) is well suited for typing of smear-positive specimens, (ii) may also be applied to gastric aspirates and other types of specimens if DNA extraction methods are modified appropriately, and (iii) exhibits a sensitivity with smear-negative sputum specimens which makes it recommendable that a minimum of three samples from the same patient be tested.     

Performance Characteristics of the BDProbeTec System for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens

Strand displacement amplification (SDA) technology has been established in a fully automated system known as BDProbeTec. Target sequences of the insertion sequence IS6110 and the 16S rRNA gene are simultaneously amplified, which thus allows the detection of Mycobacterium tuberculosis complex and, as an additional option, of most Mycobacterium species. Detection occurs via a chemiluminescent microwell assay that employs the simultaneous hybridization and capture of SDA products with a biotinylated capture probe and an alkaline phosphatase detector probe. We have evaluated the performance of the BDProbeTec system in detecting M. tuberculosis complex by testing 799 respiratory specimens and comparing the results to those obtained by conventional diagnostic techniques, i.e., microscopy and culture (solid and radiometric media). M. tuberculosis was cultivated from 41 specimens, of which 28 (68.4%) were smear positive and 13 (31.6%) were smear negative. The overall sensitivity of the SDA assay was 97.6% (for smear-positive specimens, 100%; for smear-negative specimens, 92.3%), and specificity was 95.0%. After resolution of the discrepancies by studying the patients’ clinical data, sensitivity and specificity were 97.9 and 96.5%, respectively, and positive and negative predictive values were 63.9 and 99.9%, respectively. These preliminary data demonstrate that the BDProbeTec system has promising performance characteristics with respiratory specimens and that it allows the detection of M. tuberculosis complex within hours.     

Evaluation of the Abbott LCx Mycobacterium tuberculosis Assay for Direct Detection of Mycobacterium tuberculosis Complex in Human Samples

Seven hundred thirty-seven clinical samples from 460 patients were processed for direct detection of Mycobacterium tuberculosis complex by a semiautomated ligase chain reaction commercial assay, the LCx Mycobacterium tuberculosis Assay (LCx assay) from Abbott Laboratories. Results were compared to those of direct microscopy and standard microbiological culture. Of 26 patients (5.7%) with a culture positive for M. tuberculosis, 22 (84.6%) were found positive by the LCx assay. The sensitivity of the LCx assay was 98% for smear-positive samples and 27% for smear-negative samples. With an overall culture positivity rate for M. tuberculosis of 8.3% (61 of 737 samples) and after resolution of discrepant results according to clinical data, the sensitivity, specificity, and positive and negative predictive values of the LCx assay were 78, 100, 95, and 98%, respectively, compared to 85, 100, 100, and 98%, respectively, for culture and 67, 99, 87, and 97%, respectively, for acid-fast staining. In conclusion, the LCx assay proved satisfactory and appears to be an easy-to-use 1-day test which must be used with standard culture methods but can considerably reduce diagnosis time versus culture. However, its clinical interest appears to be limited in our population with low mycobacterial prevalence because of its cost considering the small gain in sensitivity versus direct microscopy.     

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

Purpose

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed.

Materials and Methods

The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains.

Results

The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%).

Conclusion

The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.     

Humoral Immune Responses against the Mycobacterium tuberculosis 38-Kilodalton,MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis

The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging. The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in 250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and 73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.The control of tuberculosis (TB) remains challenging in China (18). Currently, the diagnosis of active TB mainly relies on clinical symptoms, radiologic findings, and the detection of Mycobacterium tuberculosis in clinical samples using smear staining and mycobacterial culture. However, the diagnosis of TB in smear- and culture-negative TB patients is difficult. The detection of M. tuberculosis-specific antibodies in human sera has been an important aid in diagnosis of TB. Notably, several antigens have been demonstrated to have merit in TB diagnosis, including the 38-kDa protein, which is commonly used in serodiagnostic tests (4, 5, 8, 13, 19, 22, 23). Previous studies suggest that the antibody responses to M. tuberculosis antigens are heterogeneous among individuals (17) so that the detection of antibodies against a single antigen usually has a low sensitivity for diagnosis of TB, especially for bacterium-negative cases. Therefore, it may be valuable to evaluate antibodies against the 38-kDa antigen and other major antigens for the diagnosis of active TB (14, 15).Notably, the MTB48, CFP-10 (culture filtrate protein 10), and ESAT-6 (6-kDa early secreted antigen target of M. tuberculosis) genes are conserved in M. tuberculosis and Mycobacterium bovis isolates but partially deleted or absent in M. bovis BCG as well as in most nontuberculous mycobacteria (NTM) (1-3, 10, 16). Importantly, the proteins encoded by these genes are immunogenic (7, 9, 12, 16). In this study, we cloned the 38-kDa, MTB48, CFP-10, and ESAT-6 genes and generated recombinant 38-kDa, MTB48, and CFP-10/ESAT-6 fusion proteins in Escherichia coli. Subsequently, we developed an enzyme-linked immunosorbent assay (ELISA) for the characterization of serum antibodies against 38-kDa, MTB48, and CFP-10/ESAT-6 antigens in a population of 250 active TB patients and 260 healthy subjects. We found that characterization of antibodies against multiple M. tuberculosis antigens were valuable for the diagnosis of active TB.     

Results from 5 Years of Nationwide DNA Fingerprinting of Mycobacterium tuberculosis Complex Isolates in a Country with a Low Incidence of M. tuberculosis Infection

Results from DNA fingerprint analyses of Mycobacterium tuberculosis complex isolates from tuberculosis (TB) patients diagnosed during 5 years in Denmark are presented. The lack of success in eradicating TB in this low-incidence country may be explained by an unrecognized high frequency of active TB transmission (57%) among native Danes. Only two strains of M. tuberculosis are responsible for 40% of all clustered cases of TB among Danes.     

Improved sensitivity of diagnosis of tuberculosis in patients in Korea via a cocktail enzyme-linked immunosorbent assay containing the abundantly expressed antigens of the K strain of Mycobacterium tuberculosis

Tuberculosis (TB) is the leading cause of death from a single infectious agent in Korea. In this study, we compared the proteins present in culture filtrates from Mycobacterium tuberculosis strain K, which is the dominant clinical isolate in Korea, with those present in culture filtrates from M. tuberculosis H37Rv. Several differences in expression were detected between the two strains for those proteins with a molecular mass of <20 kDa. ESAT-6, HSP-X, and CFP-10 were found to be abundantly expressed in the strain K culture filtrates by liquid chromatography-electrospray ionization-time of flight mass spectrometry. The serodiagnostic potentials of recombinant antigens rESAT-6, rHSP-X, and rCFP-10 and two native antigens (Ag85 and PstS1) were evaluated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera collected from 46 TB patients with active disease and 46 healthy controls. As for our ELISA results, HSP-X was superior to the other antigens in terms of sensitivity when a single antigen was employed. The results of a receiver operator characteristic analysis revealed that a cocktail ELISA using all five antigens was significantly more sensitive (77.8%) than the use of a single antigen and offered equivalent specificity; moreover, it produced the largest area under the curve (0.91 versus 0.55 to 0.87). Therefore, a cocktail ELISA containing abundantly expressed antigens enhances the sensitivity of a single antigen and can be a useful diagnostic tool for the detection of active TB.     

Identification of Mycobacterium tuberculosis Antigens of High Serodiagnostic Value

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, with several million new cases detected each year. Current methods of diagnosis are time-consuming and/or expensive or have a low level of accuracy. Therefore, new diagnostics are urgently needed to address the global tuberculosis burden and to improve control programs. Serological assays remain attractive for use in resource-limited settings because they are simple, rapid, and inexpensive and offer the possibility of detecting cases often missed by routine sputum smear microscopy. The aim of this study was to identify M. tuberculosis seroreactive antigens from a panel of 103 recombinant proteins selected as diagnostic candidates. Initial library screening by protein array analysis and enzyme-linked immunosorbent assay (ELISA) identified 42 antigens with serodiagnostic potential. Among these, 25 were novel proteins. The reactive antigens demonstrated various individual sensitivities, ranging from 12% to 78% (specificities, 76 to 100%). When the antigens were analyzed in combinations, up to 93% of antibody responders could be identified among the TB patients. Selected seroreactive proteins were used to design 3 new polyepitope fusion proteins. Characterization of these antigens by multiantigen print immunoassay (MAPIA) revealed that the vast majority of the TB patients (90%) produced antibody responses. The results confirmed that due to the remarkable variation in immune recognition patterns, an optimal multiantigen cocktail should be designed to cover the heterogeneity of antibody responses and thus achieve the highest possible test sensitivity.Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis and is one of the leading causes of mortality due to infectious disease worldwide (9). Nearly one-third of the world''s population is believed to be infected, with approximately 8.8 million new cases detected each year (30, 45). The World Health Organization (WHO) cites TB as the single most important fatal infection, with over 1.6 million deaths per year, the majority (95%) of which are in developing countries (45).Because of logistical and technical shortcomings, human TB testing in most countries is limited to clinical evaluation of symptomatic individuals and screening of high-risk populations. Compounding the severity of TB is the realization that a leading cause of death among HIV-positive people is concomitant TB, accounting for about one-third of AIDS-related deaths. It is estimated that a rapid and widely available diagnostic with 85% sensitivity and 95% specificity would result in 400,000 fewer deaths each year and would greatly reduce the global health cost of TB (18).Existing TB diagnostic methods are either too time-consuming, too complex and labor-intensive, too inaccurate, or too expensive for routine use in resource-limited settings (2, 36). For active pulmonary disease, sputum smear microscopy, culture, and/or PCR-based probes can be used to support X-ray findings and/or clinical observations suggestive of TB. Of these, microscopic examination of sputum is the only rapid, relatively simple, and inexpensive test for TB. The reported sensitivity of Ziehl-Neelsen staining of unprocessed sputum smears from immunocompetent adults is only 40 to 70% (19, 21), and it may be significantly lower for children and/or HIV-infected patients (12). A delayed or missed TB diagnosis certainly contributes to M. tuberculosis transmission and increased TB mortality (22, 27).Mycobacterial culture is the gold standard method of TB diagnosis. However, it requires up to 8 weeks for the isolation of M. tuberculosis from a clinical specimen, and importantly, in 10 to 20% of positive cases, the bacillus is not successfully cultured (3). Culture is more expensive than microscopy and requires a high standard of technical expertise. Therefore, a sensitive and specific point-of-care test for the rapid diagnosis of patients with active TB would facilitate early treatment and reduce M. tuberculosis transmission.An antibody test for TB has long been sought. Serologic assays remain attractive for use in resource-limited settings because they generally are simple, rapid, and relatively inexpensive compared to other methods. For TB, serological tests may also offer the possibility of detecting cases that are usually missed by routine sputum smear microscopy, such as extrapulmonary disease and pediatric TB. Numerous in-house serological assays for TB, using a variety of antigens to detect circulating antibodies, have been developed over the years, including complement fixation tests, hemagglutination tests, radioimmunoassays, and enzyme-linked immunosorbent assays (ELISAs) (11, 38-40). Both lateral-flow and enzyme immunoassay formats have been developed and are currently available commercially, but so far none has demonstrated adequate sensitivity and specificity (7, 13, 31, 38).In this study, we assessed a large panel of recombinant TB antigens for their serodiagnostic potential. From an initial screen of 103 recombinant proteins by protein microarray analysis and ELISA, 42 previously known and novel TB antigens were found to elicit specific antibody responses in TB patients. Several fusion proteins comprised of tandem arrangements of the selected antigens were made and serologically characterized by ELISA and multiple-antigen print immunoassays (MAPIA). The antigens identified hold promise for the development of a rapid and highly sensitive serodiagnostic test for TB.     

Clinical Evaluation of the BDProbeTec Strand Displacement Amplification Assay for Rapid Diagnosis of Tuberculosis

The reliability of the BDProbeTec MTB Test (Becton Dickinson, Sparks, Md.) for direct detection of Mycobacterium tuberculosis in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture, with the BACTEC TB 460 and Middlebrook 7H11 biplates. Patients known to have tuberculosis were excluded from analysis. Of 523 specimens from 277 patients, 53 grew a mycobacterium: 24 specimens of M. tuberculosis and 29 specimens of nontuberculous mycobacteria. After initial testing, 42 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 95.8, 96.2, 54.8, and 99.8%, respectively. After resolution of discrepancies, 28 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 100, 99.2, 85.7, and 100%, respectively. These same values were 100, 80.8, 93.4, and 100%, respectively, for smear-positive samples and 100, 99.4, 75.0, and 100%, respectively, for smear-negative specimens.     

Rapid Diagnosis of Extrapulmonary Tuberculosis by Ligase Chain Reaction Amplification

A rapid amplification-based test for the diagnosis of extrapulmonary tuberculosis, the LCx Mycobacterium tuberculosis Assay from Abbott Laboratories, was evaluated. Results from the LCx M. tuberculosis Assay were compared with those from culture and the final clinical diagnosis for each patient. A total of 526 nonrespiratory specimens from 492 patients were tested. The specimens included urine; feces; lymph node exudates; pleural, cerebrospinal, articular, and ascitic fluids; tissue biopsies; gastric aspirates; purulent exudates; blood; and bone marrow aspirates. After combination of the culture results and the patient’s clinical data, a total of 135 specimens were collected from 122 patients with a diagnosis of extrapulmonary tuberculosis. The sensitivity, specificity, and positive and negative predictive values for the LCx M. tuberculosis Assay were 77.7, 98.7, 95.2, and 93.1%, respectively; these values rose in resolved cases of TB to 78.5, 100, 100, and 93.1%, respectively. For 37 (27.4%) specimens from patients smear positive for the disease and 98 (72.6%) specimens from patients smear negative for the disease, the sensitivities of the LCx M. tuberculosis Assay were 100 and 71.1%, respectively. Statistically significant differences (P < 0.01) in sensitivities were found between culture and the LCx M. tuberculosis Assay. These differences were even greater among smear-negative specimens. The results demonstrate that the LCx M. tuberculosis Assay will provide rapid and valuable information for the diagnosis of extrapulmonary tuberculosis.     

Sensitivity of Fluorochrome Microscopy for Detection of Mycobacterium tuberculosis versus Nontuberculous Mycobacteria

The results for 6,532 consecutive mycobacterial respiratory specimens collected from 1,040 patients from 1993 to 1995 in a Texas hospital were studied to determine the sensitivity of fluorescence microscopy for detection of Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM). Smears were positive for acid-fast bacilli (AFB) in 63% (677 of 1,082) of specimens growing M. tuberculosis and 56% (638 of 1,148) of specimens growing the four most common species of NTM. Smear positivity by species was 58% (446 of 776) for M. avium complex, 51% (154 of 300) for rapidly growing mycobacteria (98% were M. abscessus), 78% (29 of 37) for M. kansasii, and 26% (9 of 35) for M. gordonae. Definite or probable disease by clinical criteria was present in 79% of patients with M. avium complex, 93% of patients with rapidly growing mycobacteria, 100% of patients with M. kansasii, and 0% of patients with M. gordonae. Patients with M. avium complex had a low incidence of AIDS (7%), and approximately 50% of non-AIDS patients had upper-lobe cavitary disease and 50% had nodular bronchiectasis. Only 23 of 6,532 (0.35%) of AFB smears were positive with a negative culture excluding patients on therapy for established mycobacterial disease. These studies suggest that NTM are as likely as M. tuberculosis to be detected by fluorescent microscopy in specimens from patients from areas endemic for NTM lung disease and at low risk for AIDS.     

Detection and Identification of Mycobacterium tuberculosis Directly from Sputum Sediments by Ligase Chain Reaction

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a ligase chain reaction (LCR)-based assay and acid-fast stain and culture techniques. Results from the LCR assay (Abbott LCx Mycobacterium tuberculosis [MTB] Assay) were compared to results from standard culture techniques held for 6 weeks. Four hundred ninety-three specimens from 205 patients suspected of pulmonary tuberculosis were included in the prospective study. Thirty-four (6.9%) of the specimens were culture positive for M. tuberculosis, and 13 (38%) of these were also fluorochrome stain positive. LCR sensitivities and specificities compared to culture were 74 and 98%, respectively. LCR sensitivity was 100% for fluorochrome stain-positive specimens and 57% for fluorochrome stain-negative specimens. Nine LCR-negative, culture-positive specimens were the result of low concentrations of M. tuberculosis. No inhibitors were detected in any of these specimens. Of the eight LCR-positive, culture-negative specimens, five were from patients with active tuberculosis. With these considered culture misses, final LCR sensitivity, specificity, positive predictive value, and negative predictive value were 77, 99, 91, and 98%, respectively. The same performance values for the fluorochrome acid-fast bacillus smear were 33, 98, 62, and 94%, respectively. After normal laboratory sputum processing, the Abbott LCx MTB Assay can be completed in 6 h. Thus, it is possible to have results available within 8 h of specimen submission.     

Comparative Evaluation of Initial and New Versions of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for Direct Detection of Mycobacterium tuberculosis in Respiratory and Nonrespiratory Specimens

We evaluated the initial version of the Amplified Mycobacterium Tuberculosis Direct Test (Gen-Probe) (AMTDT 1) and the new version of AMTDT (AMTDT 2) for the detection of Mycobacterium tuberculosis directly from respiratory and nonrespiratory samples and compared the results with those of culture and staining methods. The assays were applied to 410 respiratory and 272 nonrespiratory samples collected from 515 patients. The combination of the culture results and clinical diagnosis was considered to be the “gold standard.” Ninety-five respiratory specimens were collected from 67 patients with a diagnosis of pulmonary tuberculosis (TB) and 68 nonrespiratory specimens were collected from 61 patients with a diagnosis of extrapulmonary TB. With respiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 96%, respectively, for AMTDT 1 and 94.7, 100, 100, and 98.4%, respectively, for AMTDT 2. With nonrespiratory specimens, the sensitivity, specificity, and positive and negative predictive values were 83, 100, 100, and 94%, respectively, for AMTDT 1 and 86.8, 100, 100, and 98.4%, respectively, for AMTDT 2. The overall results of AMTDT 1 and AMTDT 2 were concordant for 97% (661 of 682) of the samples. Statistically significant differences in sensitivities were found between AMTDT 1 and AMTDT 2 with respiratory specimens. It was concluded that although both nucleic acid amplification methods are rapid, sensitive, and specific for the detection of M. tuberculosis complex in all types of clinical samples, AMTDT 2 appeared to be more sensitive than AMTDT 1 when applied to smear-negative specimens. In contrast AMTDT 2 is more susceptible than AMTDT 1 to inhibitory substances in the amplification reaction. The turnaround time of AMTDT 2 is shorter (3.5 h) than that for AMTDT 1 (5 h).     

Direct Detection of Mycobacterial Species in Pulmonary Specimens by Two Rapid Amplification Tests,the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test and the GenoType Mycobacteria Direct Test

Nucleic acid amplification tests have improved tuberculosis diagnostics considerably. This study evaluates a new amplification test, the GenoType Mycobacteria Direct (GTMD) test, for detection of the Mycobacterium tuberculosis complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium malmoense directly in 61 sputum samples. Thirty (49.2%) samples were auramine smear positive, and 31 (50.8%) were smear negative. The GTMD results were compared to the Gen-Probe Amplified M. tuberculosis Direct (MTD) test results, using culturing and sequencing of the 16S rRNA gene as reference methods. The GTMD test could identify 28 of 29 samples containing the M. tuberculosis complex and was negative in a sputum sample containing M. intracellulare. The overall sensitivity and specificity results were 93.3% and 90.0% for the GTMD test, respectively, and 93.1% and 93.5% for the MTD test, respectively. The GTMD test is rapid and can be easily included in routine clinical laboratories for the direct detection of the M. tuberculosis complex in smear-positive sputum samples as an adjunct to microscopy and culture. Further studies are needed to evaluate the performance of the GTMD test for the detection of atypical mycobacteria.Worldwide, tuberculosis (TB) is a major cause of illness and death. WHO estimates that in 2006, 9.2 million new cases and 1.7 million deaths occurred from TB globally (25), and the incidence is increasing. The emergence of multidrug-resistant TB, and recently also extensively drug-resistant TB, and the human immunodeficiency virus-TB coinfection are further worsening the situation, and effort to accelerate progress in global TB control is needed. Important factors for TB control are increased case detection and treatment success rates (25). The slow growth of most pathogenic mycobacteria results in diagnosis and treatment delay and has stimulated the development of nucleic acid amplification (NAA) tests for identification of mycobacteria directly in clinical specimens. NAA tests provide test results within 1 day. In general, the specificity result for NAA tests ranges from 95% to 100% (1, 12, 16, 23), but the sensitivity result, especially for acid-fast bacillus (AFB) smear-negative samples, varies greatly, from 33 to 96% (1, 12, 16, 23). For AFB smear-positive respiratory specimens, the sensitivity level is approximately 95%.Two direct systems approved by the United States Food and Drug Administration (FDA) for detection of pulmonary TB are commercially available, as follows: the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic Systems, Indianapolis, IN) and the Gen-Probe Amplified M. tuberculosis Direct test (MTD test; Gen-Probe, San Diego, CA). Both tests use the 16S rRNA gene as the target amplification gene. The 16S rRNA gene represents a stable property of microorganisms and is widely used as the target for identifying mycobacterium species. Several studies have confirmed an excellent test proficiency (sensitivity and specificity levels of more than 95%) in AFB smear-positive sputum samples but a reduced sensitivity level (82 to 85%) when applied on AFB smear-negative samples (1, 16, 23, 24). Thus, their use was limited to respiratory smear-positive samples from untreated patients. An enhanced version of the MTD test was later approved for use in both smear-positive and smear-negative specimens (5). A novel, commercially available NAA test for diagnosis of TB directly in patient specimens which has not yet been FDA approved is the BD ProbeTec ET test (Becton Dickinson Diagnostic Systems, Sparks, MD). The test is based on strand-displacement amplification of target sequences in IS6110 and the 16S rRNA gene and has a sensitivity level of 90 to 100% and a specificity level of 92% in smear-positive sputum samples (16). To make the NAA tests more rapid, robust, and applicable in laboratories without substantial technical infrastructure, the following novel NAA tests have been developed: the loop-mediated isothermal amplification (LAMP) test (Eiken Chemical Co., Ltd., Tokyo, Japan) (2, 3), the GeneXpert system (Cepheid, Sunnyvale, CA) (9), and the gold nanoparticle probes assay (21). Simple sample processing, amplification, and detection steps make these NAA tests more applicable in low-income countries with high incidence of TB. However, data on test proficiencies are limited so far. Ongoing studies will show if these rapid molecular tests can be alternatives to the conventional TB diagnostic tests.Recently, a new DNA strip test for detection of mycobacteria directly in smear-positive and smear-negative respiratory samples has been developed. The GenoType Mycobacteria Direct (GTMD) test (Hain Lifescience GmbH, Nehren, Germany) is based on nucleic acid sequence-based amplification and amplifies single-stranded nucleic acids from the 23S rRNA gene in an isothermal reaction. The biotinylated amplified DNA product is hybridized to specific oligonucleotide probes immobilized on the strip. The GTMD test detects members of the M. tuberculosis complex (MTC), Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium malmoense directly from decontaminated respiratory specimens, and the result is available within 1 day. Few studies have previously evaluated the GTMD test (7, 15, 20).The aim of this study was to evaluate the performance of the GTMD test and compare that test to the MTD test. Thus, the GTMD and MTD tests were evaluated for sensitivity and specificity using 61 respiratory specimens from patients suspected to suffer from pulmonary TB. Amplification and sequencing of the 16S rRNA gene of strains isolated from specimen culture (solid and automated liquid media) were used as reference methods.     

Evaluation of an Immunochromatographic Assay Kit for Rapid Identification of Mycobacterium tuberculosis Complex in Clinical Isolates

We evaluated a new immunochromatographic assay (ICA) using mouse monoclonal anti-MPT64 antibody for rapid discrimination between Mycobacterium tuberculosis and nontuberculous mycobacteria in clinical isolates. A study with mycobacteria and other organisms showed excellent sensitivity (99%) and specificity (100%) and an appropriate detection limit (10⁵ CFU/ml) when tested with M. tuberculosis H37Rv. This ICA can simplify the identification of M. tuberculosis in clinical laboratories.     

Value of Examining Multiple Sputum Specimens in the Diagnosis of Pulmonary Tuberculosis

To objectively assess the value of examining multiple sputum specimens in maximizing the sensitivity of detection of Mycobacterium tuberculosis, we retrospectively reviewed the acid-fast bacillus smear and culture results of patients diagnosed with culture-proven pulmonary tuberculosis (TB) at Hennepin County Medical Center between 1986 and 1996. Two hundred and forty six persons were diagnosed with pulmonary TB in the time period analyzed. In 93% of these cases (229 of 246) the laboratory diagnosis was made by detection of M. tuberculosis in sputum specimens; however, only 52% (120 of 229) of these patients had at least three sputum specimens submitted to the laboratory at the time of diagnosis. Of the patients from whom at least three specimens were collected, 47% (56 of 120) had at least one smear-positive specimen; the third or later specimen submitted was the first smear-positive specimen for 13% (7 of 56) of these persons but was the first culture-positive specimen for only 7% (4 of 56). Of the 64 patients with smear-negative specimens, for only 5% (3 of 64) was the third or subsequent specimen submitted the first from which M. tuberculosis was recovered. This data indicates that, in our institution, the overwhelming majority of culture-proven pulmonary TB cases are diagnosed from the first or second sputum specimen submitted to the laboratory and that only rarely is a third specimen of diagnostic value.     

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 μg 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.     

Development of multiplex assay for rapid characterization of Mycobacterium tuberculosis

We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB, katG, inhA, embB), members of the M. tuberculosis complex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG, gyrA), and Haarlem and Beijing strains (ogt, mutT2, mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.     

Clinical Evaluation of the Gen-Probe Amplified Direct Test for Detection of Mycobacterium tuberculosis Complex Organisms in Cerebrospinal Fluid

Eighty-four cerebrospinal fluid (CSF) samples from different children who presented with signs and symptoms of meningitis were evaluated for the presence of Mycobacterium tuberculosis complex organisms by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe, San Diego, Calif.). All CSF samples had negative acid-fast smears by the Ziehl-Neelsen staining method. M. tuberculosis was recovered from five samples. M. tuberculosis did not grow from 19 additional samples, but the samples were from patients who fulfilled specific clinical and laboratory criteria for probable tuberculous meningitis (TBM). The remaining samples (n = 60) were from patients with other infections or noninfectious causes of meningitis. The results of the MTD were interpreted as positive or negative on the basis of recommended cutoff values for respiratory specimens. These results were interpreted as true or false positives or true or false negatives on the basis of the results of M. tuberculosis culture or whether the patient fulfilled criteria for probable TBM. The Gen-Probe MTD was 33% sensitive and 100% specific for detecting M. tuberculosis complex organisms in these 84 CSF samples. If the cutoff values for positive results were decreased for the MTD (≥11,000 versus ≥30,000 relative light units), the sensitivity increased to 83% and the specificity remained 100%. These results for the MTD are encouraging considering that TBM is a highly fatal disease and difficult to diagnose by conventional laboratory techniques.