Clinical evaluation of the polymerase chain reaction for the rapid diagnosis of tuberculosis

AIMS: Use of the polymerase chain reaction for the detection of Mycobacterium tuberculosis (TB PCR) as a basis for making clinical decisions on the initiation of antituberculosis treatment was studied. METHODS: A retrospective study involving a cohort of 155 patients being investigated for tuberculosis in an infectious disease consultation service was undertaken. TB PCR was performed on pulmonary and extrapulmonary specimens from these patients. The sensitivity of TB PCR was analysed. RESULTS: Of the 155 patients, 144 fitted the clinical diagnosis of tuberculosis, and 112 of them were culture positive for M tuberculosis. Sixty (58.3%) patients with clinical features suggestive of tuberculosis received antituberculosis treatment based on positive TB PCR alone. Of 224 clinical specimens (138 pulmonary and 86 extrapulmonary) sent for TB PCR, 148 (99 pulmonary and 49 extrapulmonary) were positive in 117 patients. Of the 690 clinical specimens sent for culture, 279 were positive for M tuberculosis in 112 patients. The diagnostic sensitivity of TB PCR was 75.9% (85 of 112) and 81.3% (117 of 144) in patients with culture confirmed and clinically diagnosed tuberculosis, respectively. Using culture as the gold standard, the overall sensitivity of TB PCR was 78.3%, and for pulmonary and extrapulmonary specimens it was 82.3% and 72.0%, respectively. CONCLUSIONS: TB PCR is a rapid and reliable test in the diagnosis and management of tuberculosis.     

Molecular methods in the detection and identification of mycobacterial infections.

Nucleic acid amplification (NAA) tests for direct detection of Mycobacterium tuberculosis complex in respiratory specimens have the potential to provide a more rapid diagnosis of pulmonary tuberculosis (TB) than is currently possible by conventional stain, culture, and identification tests. Currently, 2 NAA tests-enhanced Amplified Mycobacterium Tuberculosis Direct (MTD) Test (Gen-Probe, Inc) and Amplicor Mycobacterium tuberculosis Test (Roche Molecular Systems, Inc)-have been approved by the Food and Drug Administration for testing respiratory specimens that are smear positive for acid-fast bacilli (AFB). This restriction to AFB smear-positive specimens was based on data from the initial clinical trials conducted to evaluate these products that showed low sensitivity (ie, 48%-53%) and less-than-optimal specificity (ie, 96%-99%) in AFB smear-negative specimens. Data from the clinical trial for the enhanced MTD test and from 2 subsequent studies, however, suggest that this version of the MTD test is a reliable tool for rapid diagnosis of pulmonary TB, regardless of the AFB smear result. Both NAA tests have been evaluated for diagnosis of extrapulmonary TB, and results were comparable to the results of tests performed with respiratory specimens. The NAA tests also appear to be reliable for rapid identification of M tuberculosis complex in positive broth cultures of all specimen types except blood. The impact of the NAA tests on patient outcome varies based on the AFB smear result. With smear-positive results, public health and hospital infection control resources are predominantly affected. With smear-negative results, however, the potential for affecting patient outcome is much greater. In patients with smear-negative results, the NAA test can result in earlier diagnosis of TB and subsequent initiation of therapy. Use of these tests also may eliminate the need for invasive diagnostic procedures, which are costly and pose an added risk to the patient, and they may allow earlier discharge of hospitalized patients.     

Immunochromatographic IgG/IgM test for rapid diagnosis of active tuberculosis

For rapid diagnosis and discrimination between active tuberculosis (TB) and other pulmonary diseases, we evaluated the clinical usefulness of detection of serum immunoglobulin IgG and IgM antibodies raised against mycobacterial 38-kDa, 16-kDa, and 6-kDa antigens by a commercial rapid immunochromatographic IgG/IgM test (Standard Diagnostics, South Korea) in 246 serum samples from three groups of patients: (i) 171 patients with active TB (128 with pulmonary TB [pTB] and 43 with extrapulmonary TB [epTB]), (ii) 73 patients with pulmonary non-TB diseases, and (iii) two leprosy patients. The sensitivities of IgG and IgM in patients with active TB (pTB and epTB) were 68.4% and 2.3%, respectively. IgG had the best performance characteristics, with sensitivities of 78.1% and 39.5% in sera from patients with active pTB and epTB, respectively, and a specificity of 100%. The sensitivities of IgM were poor and were similar for pTB and epTB (2.3%). In contrast, specificity was very elevated (100%). The combination of IgG with IgM did not improve its sensitivity. IgG-mediated responses against the mycobacterial 38-kDa, 16-kDa, and 6-kDa antigens might constitute a clinically useful tool for presumptive diagnosis and discrimination of active pTB from other pulmonary diseases. Moreover, based on its simplicity and rapidity of application, it could be a screening tool for active pTB in poorly equipped laboratories.     

Laboratory reporting of tuberculosis test results and patient treatment initiation in California

Prompt laboratory reporting of tuberculosis (TB) test results is necessary for TB control. To understand the extent of and factors contributing to laboratory reporting delays and the impact of reporting delays on initiation of treatment of TB patients, we analyzed data from 300 consecutive culture-positive TB cases reported in four California counties in 1998. Laboratory reporting to the specimen submitter was delayed for 26.9% of smear-positive patients and 46.8% of smear-negative patients. Delays were associated with the type of laboratory that performed the testing and with delayed transport of specimens. Referral laboratories (public health and commercial) had longer median reporting time frames than hospital and health maintenance organization laboratories. Among patients whose treatment was not started until specimens were collected, those with delayed laboratory reporting were more likely to have delayed treatment than patients with no laboratory reporting delays (odds ratio [OR] of 3.9 and 95% confidence interval [CI] of 1.6 to 9.7 for smear-positive patients and OR of 13.1 and CI of 5.3 to 32.2 for smear-negative patients). This relation remained after adjustment in a multivariate model for other factors associated with treatment delays (adjusted OR of 25.64 and CI of 7.81 to 83.33 for smear-negative patients). These findings emphasize the need to reduce times of specimen transfer between institutions and to ensure rapid communication among laboratories, health care providers, and health departments serving TB patients.     

Evaluation of commercial enzyme-linked immunosorbent assay kits for detection of tuberculosis in Argentinean population

Pathozyme-Myco G (Myco G), M, A, and TB complex plus (Omega Diagnostics Ltd., Alloa, Scotland) were evaluated for the serological diagnosis of pulmonary tuberculosis (TB) in an Argentinean population. Sera from 58 patients with pulmonary TB, 24 subjects with pulmonary mycobacteriosis or mycoses (pulmonary MM group), and 45 subjects with other underlying disorders (control group) were analyzed. The sensitivities of the tests ranged from 29% (Myco M) to 82% (Myco G) in smear-positive patients (17 subjects) and from 29% (TB complex plus) to 49% (Myco G) in smear-negative patients (41 subjects). The specificities of the assays varied from 93% (Myco M) to 100% (Myco G and TB complex plus) in controls and from 62% (Myco A) to 96% (TB complex plus) in the pulmonary MM group. Overall, for the diagnosis of smear-negative patients, Myco G had the best characteristics, with a sensitivity of 49% and specificities of 100% for controls and 75% for the pulmonary MM group; after its combination with TB complex plus, its sensitivity improved to 59%. Nevertheless, despite its relatively poor capacity to discriminate between pulmonary TB and pulmonary MM, Myco G, alone or in combination with TB complex plus, would be a useful diagnostic tool for patients with suspected pulmonary TB living in areas where the relative prevalence of pulmonary MM was low.     

Evaluation of a monoclonal antibody (TB72) based serological test for tuberculosis.

A serological test for tuberculosis (TB), based on competitive inhibition by human sera of antigen binding by a murine monoclonal antibody (TB72) has been performed using solid phase radioimmunoassay. The test was evaluated on sera from patients with pulmonary or extra-pulmonary active TB as well as from control hospitalized patients--half of whom had various chest complaints. Positive values were found in 74% of all or 55% of untreated active pulmonary TB. Patients with extrapulmonary TB segregated, whereby antibody titres were increased in cases of pleural, peritoneal, pericardial or bone infection but only marginal or negative values were found in the majority of patients with lymphatic and genitourinary infection. None of the subjects with suspected but excluded tuberculosis or sarcoidosis had demonstrable TB72 antibodies. Generally there was a positive correlation between the levels of TB72 like and 'total' Mycobacterium tuberculosis sonicate binding antibodies. In particular, the titre of sonicate binding antibodies did not exceed background levels in patients with active pulmonary tuberculosis who were negative in the TB72 competition test. None of these false negative patients received drug therapy whereas most patients with the highest antibody levels were under chemotherapy for 1-8 months prior to serum testing. The serodiagnostic value of the TB72 based competition test has been discussed.     

Exploring serological classification tree model of active pulmonary tuberculosis by magnetic beads pretreatment and MALDI-TOF MS analysis

Pulmonary tuberculosis (TB) is an infectious disease disturbing status of public health, and accurate diagnosis of TB would effectively help control the disturbance. Our study tried to establish a classification tree model that distinguished active TB from non-TB individuals. We used matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) combined with weak cationic exchange (WCX) magnetic beads to analyse 178 serum samples containing 75 patients with active TB and 103 non-TB individuals (43 patients with common pulmonary diseases and 60 healthy controls). Samples were randomly divided into a training set and a test set. Statistical softwares were applied to construct this model. An amount of 48 differential expressed peaks (P < 0.05) were identified by the training set, and our model was set up by three of them, m/z 7626, 8561 and 8608. This model can discriminate patients with active TB from patients with non-TB with a sensitivity of 98.3% and a specificity of 84.4%. The test set was used to verify the performance, which demonstrated good sensitivity and specificity: 85.7% and 83.3%, respectively. Differential expressed peaks between smear-positive and smear-negative active TB also have been analysed. It came out that m/z 8561 and 8608 not only acted as vital factors in the pathogenesis of active TB but also played an important role in regulating different active TB status. In conclusion, MALDI-TOF MS combined with WCX magnetic beads was a powerful technology for constructing classification tree model, and the model we built could serve as a potential diagnostic tool for active TB.     

Use of Transrenal DNA for the Diagnosis of Extrapulmonary Tuberculosis in Children: a Case of Tubercular Otitis Media

The diagnosis of tuberculosis (TB) is difficult in children, especially for smear-negative pulmonary and extrapulmonary TB, which are common at this age. We report an 11-year-old girl with TB otitis media with negative smear microscopy and Xpert MTB/RIF but positive Mycobacterium tuberculosis-specific transrenal DNA (Tr-MTB-DNA) test results and culture for M. tuberculosis.     

Clinical evaluation of the enhanced Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for rapid diagnosis of tuberculosis in prison inmates

The reliability of the enhanced Amplified Mycobacterium Tuberculosis Direct Test (E-MTD; Gen-Probe, Inc., San Diego, Calif.) for rapid diagnosis of pulmonary tuberculosis (TB) was evaluated by testing 1, 004 respiratory specimens from 489 Texas prison inmates. Results were compared to those of mycobacterial culture (BACTEC TB 460 and Middlebrook 7H11 biplates), smear for acid-fast bacilli (AFB; auramine O), and clinical course. After chart review, three patients (nine specimens) who were on antituberculosis therapy before the study began were excluded from final analysis. Of the remaining 995 specimens, 21 were AFB smear positive: 13 grew Mycobacterium tuberculosis complex (MTBC), 6 grew nontuberculous mycobacteria, and 2 (from two patients diagnosed with TB and started on therapy after the study began) were culture negative. Twenty-eight specimens (20 patients) were positive for MTBC by culture and E-MTD. Seven specimens (seven patients) were positive by culture alone; three were from patients who had other E-MTD-positive specimens, two were false-positive cultures, and two were false-negative E-MTD results. Eight specimens were positive by E-MTD only; four specimens (four patients) were false-positive E-MTD results, and four specimens were from two patients with earlier E-MTD-positive specimens that grew MTBC. Thus, there were 22 patients with TB (10 smear positive and 12 smear negative). The sensitivity and specificity of the AFB smear for diagnosis of TB, by patient, were 45.5 and 98.9%, respectively. After resolving discrepancies, these same values for E-MTD were 90.9 and 99.1% overall, 100 and 100% for the smear-positive patients, and 83.3 and 99.1% for the smear-negative patients. Excluding the one smear-negative patient whose E-MTD-negative, MTBC culture-positive specimen contained inhibitory substances, the sensitivity of E-MTD was 95.2% overall and 90.9% in smear-negative patients. The specificity and positive predictive value of E-MTD can be improved, without altering other performance characteristics, by modifying the equivocal zone recommended by the manufacturer. These data suggest that E-MTD is a reliable method for rapid diagnosis of pulmonary TB, irrespective of the AFB smear result. Guidelines for the most appropriate use of E-MTD with smear-negative patients are needed.     

Association of strong immune responses to PPE protein Rv1168c with active tuberculosis

Accurate diagnosis of tuberculosis (TB) infection is critical for the treatment, prevention, and control of TB. Conventional diagnostic tests based on purified protein derivative (PPD) do not achieve the required diagnostic sensitivity. Therefore, in this study, we have evaluated the immunogenic properties of Rv1168c, a member of the PPE family, in comparison with PPD, which is routinely used in the tuberculin test, and Hsp60 and ESAT-6, well-known immunodominant antigens of Mycobacterium tuberculosis. In a conventional enzyme immunoassay, the recombinant Rv1168c protein displayed stronger immunoreactivity against the sera obtained from patients with clinically active TB than did PPD, Hsp60, or ESAT-6 and could distinguish TB patients from Mycobacterium bovis BCG-vaccinated controls. Interestingly, Rv1168c antigen permits diagnosis of smear-negative pulmonary TB as well as extrapulmonary TB cases, which are often difficult to diagnose by conventional tests. The immunodominant nature of Rv1168c makes it a promising candidate to use in serodiagnosis of TB. In addition, our studies also show that Rv1168c is a potent T-cell antigen which elicits a strong gamma interferon response in sensitized peripheral blood mononuclear cells obtained from TB patients.     

Diagnostic value of bronchoalveolar lavage and bronchial washing in sputum-scarce or smear-negative cases with suspected pulmonary tuberculosis: a randomized study

ObjectivesBronchoalveolar lavage (BAL) and bronchial washing (BW) are two major methods used to obtain high-quality respiratory specimens from patients with suspected pulmonary tuberculosis (TB) but a sputum-scarce or smear-negative status. We aimed to compare the value of BAL and BW in the diagnosis of TB in such patients.MethodsWe enrolled patients with suspected pulmonary TB but with a sputum-scarce or smear-negative status who were referred for bronchoscopy between October 2013 and January 2016. Participants were randomized into the BAL and BW groups for evaluation. The primary outcome was the diagnostic yield for TB detection. Secondary outcomes included culture positivity, positivity of nucleic acid amplification tests (NAATs) for Mycobacterium tuberculosis and procedure-related complications.ResultsA total of 94 patients were assessed and 91 (43 in the BAL group, 48 in the BW group) were analysed. Twenty-one patients (48.8%) in the BAL group and 30 (62.5%) in the BW group had a final diagnosis of pulmonary TB. The detection rate of M. tuberculosis by culture or NAAT was significantly higher in BAL specimens than in BW specimens (85.7% vs 50.0%, p 0.009). The procedure-related complications were hypoxic events, 2/43 (4.7%) in the BAL group and 5/48 (10.4%) in the BW group; and post-bronchoscopic fever, 3/43 (7.0%) in the BAL group and 4/48 (8.3%) in the BW group.DiscussionAs long as it is tolerable, BAL rather than BW, should be used to obtain specimens for the diagnosis of pulmonary TB in sputum-scarce or smear-negative cases.     

Evaluation of Antigen-Specific Immunoglobulin G Responses in Pulmonary Tuberculosis Patients and Contacts

This study aimed to evaluate the serodiagnostic potential of immunoglobulin G (IgG) responses to Mycobacterium tuberculosis antigens in pulmonary tuberculosis (TB) patients, recent TB contacts with latent TB infection (LTBI), and healthy subjects. Infections were assessed using tuberculin skin tests, QuantiFERON-TB Gold In-Tube tests, drug susceptibility testing, and molecular genotyping of clinical isolates. Serum IgG responses to selective M. tuberculosis antigens, including the 38-kDa and 16-kDa antigens, lipoarabinomannan (LAM), and recombinant early secreted antigen target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10), were determined. We found that the serum IgG responses to all antigens might differentiate between active TB and LTBI, with LAM having the highest diagnostic value (area under the curve [AUC] of 0.7756, P < 0.001). Recurrent TB cases showed significantly higher IgG responses to 38 kDa, CFP-10 (P < 0.01), and LAM (P < 0.05) than new cases, and male patients had higher levels of antigen-specific IgG than females (P < 0.05). Conversely, drug resistance and patient body mass index did not affect IgG responses (P > 0.05). LAM-specific IgG responses differentiated between acid-fast bacillus (AFB) smear-positive and -negative patients (P < 0.01), whereas antigen-specific IgG responses did not vary with the M. tuberculosis genotype (P > 0.05). Significantly higher IgG responses to 38 kDa and 16 kDa were observed in AFB smear-negative patients than in controls. These results suggest that assessment of serum IgG responses to selective purified M. tuberculosis antigens may help improve the diagnosis of active TB, particularly for sputum smear-negative patients or recurrent cases, and these may also help to differentiate between active TB and LTBI.     

Comparison of the Xpert MTB/RIF test with an IS6110-TaqMan real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens

The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.     

Xpert MTB/RIF: a New Pillar in Diagnosis of Extrapulmonary Tuberculosis?

Approximately 10 to 15% of tuberculosis (TB) cases in India are estimated to have extrapulmonary disease, and due to a lack of diagnostic means, they often remain untreated. The early detection of Mycobacterium tuberculosis and multidrug resistance is a priority in TB diagnosis to improve the successful treatment rate of TB and reduce transmission. The Xpert MTB/RIF (Xpert) test, recently endorsed by the World Health Organization for the detection of pulmonary TB, was evaluated to test its utility in 547 patients with suspected extrapulmonary tuberculosis. Five hundred forty-seven extrapulmonary specimens were split and processed simultaneously for both culture (solid and liquid) and Xpert testing. For culture, the sensitivity was low, 53% (150/283 specimens). Xpert sensitivity and specificity results were assessed in comparison to a composite reference standard made up of smear and culture results and clinical, radiological, and histological findings. The sensitivity of the Xpert assay was 81% (228/283 specimens) (64% [89/138] for smear-negative cases and 96% [139/145] for smear-positive cases), with a specificity of 99.6%. The sensitivity was found to be high for the majority of specimen types (63 to 100%) except for cerebrospinal fluid, the sensitivity of which was 29% (2/7 specimens). The Xpert test correctly identified 98% of phenotypic rifampin (RIF)-resistant cases and 94% of phenotypic RIF-susceptible cases. Sequencing of the 6 discrepant samples resolved 3 of them, resulting in an increased specificity of 98%. In conclusion, the results of this study suggest that the Xpert test also shows good potential for the diagnosis of extrapulmonary TB and that its ease of use makes it applicable for countries where TB is endemic.     

Experimental platform utilising melting curve technology for detection of mutations in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates

Tuberculosis (TB) remains one of the most deadly infections with approximately a quarter of cases not being identified and/or treated mainly due to a lack of resources. Rapid detection of TB or drug-resistant TB enables timely adequate treatment and is a cornerstone of effective TB management. We evaluated the analytical performance of a single-tube assay for multidrug-resistant TB (MDR-TB) on an experimental platform utilising RT-PCR and melting curve analysis that could potentially be operated as a point-of-care (PoC) test in resource-constrained settings with a high burden of TB. Firstly, we developed and evaluated the prototype MDR-TB assay using specimens extracted from well-characterised TB isolates with a variety of distinct rifampicin and isoniazid resistance conferring mutations and nontuberculous Mycobacteria (NTM) strains. Secondly, we validated the experimental platform using 98 clinical sputum samples from pulmonary TB patients collected in high MDR-TB settings. The sensitivity of the platform for TB detection in clinical specimens was 75% for smear-negative and 92.6% for smear-positive sputum samples. The sensitivity of detection for rifampicin and isoniazid resistance was 88.9 and 96.0% and specificity was 87.5 and 100%, respectively. Observed limitations in sensitivity and specificity could be resolved by adjusting the sample preparation methodology and melting curve recognition algorithm. Overall technology could be considered a promising PoC methodology especially in resource-constrained settings based on its combined accuracy, convenience, simplicity, speed, and cost characteristics.     

Diagnosis of pulmonary tuberculosis in HIV-positive patients by microscopic observation drug susceptibility assay

The microscopic observation drug susceptibility assay (MODS) is a novel and promising test for the early diagnosis of tuberculosis (TB). We evaluated the MODS assay for the early diagnosis of TB in HIV-positive patients presenting to Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases in southern Vietnam. A total of 738 consecutive sputum samples collected from 307 HIV-positive individuals suspected of TB were tested by smear, MODS, and the mycobacteria growth indicator tube method (MGIT). The diagnostic sensitivity and specificity of MODS compared to the microbiological gold standard (either smear or MGIT) were 87 and 93%, respectively. The sensitivities of smear, MODS, and MGIT were 57, 71, and 75%, respectively, against clinical gold standard (MODS versus smear, P<0.001; MODS versus MGIT, P=0.03). The clinical gold standard was defined as patients who had a clinical examination and treatment consistent with TB, with or without microbiological confirmation. For the diagnosis of smear-negative patients, the sensitivities of MODS and MGIT were 38 and 45%, respectively (P=0.08). The median times to detection using MODS and MGIT were 8 and 11 days, respectively, and they were 11 and 17 days, respectively, for smear-negative samples. The original bacterial/fungal contamination rate of MODS was 1.1%, while it was 2.6% for MGIT. The cross-contamination rate of MODS was 4.7%. In conclusion, MODS is a sensitive, specific, and rapid test that is appropriate for the detection of HIV-associated TB; its cost and ease of use make it particularly useful in resource-limited settings.     

Molecular detection and identification of mycobacterium tuberculosis complex and four clinically important nontuberculous mycobacterial species in smear-negative clinical samples by the genotype mycobacteria direct test

Although the sensitivity and specificity of nucleic acid amplification assays are high with smear-positive samples, the sensitivity with smear-negative and extrapulmonary samples for the diagnosis of tuberculosis in suspicious tuberculosis cases still remains to be investigated. This study evaluates the performance of the GenoType Mycobacteria Direct (GTMD) test for rapid molecular detection and identification of the Mycobacterium tuberculosis complex and four clinically important nontuberculous mycobacteria (M. avium, M. intracellulare, M. kansasii, and M. malmoense) in smear-negative samples. A total of 1,570 samples (1,103 bronchial aspiration, 127 sputum, and 340 extrapulmonary samples) were analyzed. When we evaluated the performance criteria in combination with a positive culture result and/or the clinical outcome of the patients, the overall sensitivity, specificity, and positive and negative predictive values were found to be 62.4, 99.5, 95.9, and 93.9%, respectively, whereas they were 63.2, 99.4, 95.7, and 92.8%, respectively, for pulmonary samples and 52.9, 100, 100, and 97.6%, respectively, for extrapulmonary samples. Among the culture-positive samples which had Mycobacterium species detectable by the GTMD test, three samples were identified to be M. intracellulare and one sample was identified to be M. avium. However, five M. intracellulare samples and an M. kansasii sample could not be identified by the molecular test and were found to be negative. The GTMD test has been a reliable, practical, and easy tool for rapid diagnosis of smear-negative pulmonary and extrapulmonary tuberculosis so that effective precautions may be taken and appropriate treatment may be initiated. However, the low sensitivity level should be considered in the differentiation of suspected tuberculosis and some other clinical condition until the culture result is found to be negative and a true picture of the clinical outcome is obtained.     

Evaluation of the GeneXpert MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in pulmonary and extrapulmonary specimens

Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. The rapid diagnosis of tuberculosis and detection of rifampin (RIF) resistance are essential for early disease management. The GeneXpert MTB/RIF assay is a novel integrated diagnostic device for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens. We determined the performance of the MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in smear-positive and smear-negative pulmonary and extrapulmonary specimens obtained from possible tuberculosis patients. Two hundred fifty-three pulmonary and 176 extrapulmonary specimens obtained from 429 patients were included in the study. One hundred ten (89 culture positive and 21 culture negative for M. tuberculosis) of the 429 patients were considered to have tuberculosis. In pulmonary specimens, sensitivities were 100% (27/27) and 68.6% (24/35) for smear-positive and smear-negative specimens, respectively. It had a lower sensitivity with extrapulmonary specimens: 100% for smear-positive specimens (4/4) and 47.7% for smear-negative specimens (21/44). The test accurately detected the absence of tuberculosis in all 319 patients without tuberculosis studied. The MTB/RIF assay also detected 1 RIF-resistant specimen and 88 RIF-susceptible specimens, and the results were confirmed by drug susceptibility testing. We concluded that the MTB/RIF test is a simple method, and routine staff with minimal training can use the system. The test appeared to be as sensitive as culture with smear-positive specimens but less sensitive with smear-negative pulmonary and extrapulmonary specimens that include low numbers of bacilli.     

Potential Role of Immunodiagnosis for Pulmonary Tuberculosis Using Induced Sputum Cells

PurposeTo evaluate the diagnostic utility and predictors for determinate results of an enzyme-linked immunospot assay using induced sputum cells (IS ELISPOT) for a rapid diagnosis of pulmonary tuberculosis (TB).ResultsA total of 43 subjects, including 25 with TB (TB group) and 18 with non-TB disease (non-TB group) were enrolled. Results of IS ELISPOT were determinate in only 17/43 (39%) subjects, but all of determinate results were consistent with the final diagnosis. Of the 43 sputum samples, 11 (26%) were inadequate to perform IS ELISPOT. Of 32 adequate sputum samples, the proportion of determinate results was significantly higher in the TB group (75%, 15/20) than in the non-TB group (17%, 2/12) (p=0.002). The status of active TB was a unique predictor but smear positivity was not a significant predictor for determinate results. In addition, sensitivity of IS ELISPOT (75%, 9/12) in smear negative TB was higher than that of TB-polymerase chain reaction (25%, 3/12).ConclusionIS ELISPOT showed relatively high diagnostic value and accuracy in the TB group, independent of smear positivity. IS ELISPOT may provide additional diagnostic yield for microbiological tools in the rapid diagnosis of smear-negative TB.     

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.