Immunoblot analysis for serodiagnosis of tuberculosis using a 45/47-kilodalton antigen complex of Mycobacterium tuberculosis.

We evaluated the immunoglobulin G (IgG) antibody response to the 45/47-kDa secreted protein of Mycobacterium tuberculosis by immunoblot assay, to assess its potential value for serological diagnosis. Control subjects consisted of healthy volunteers with negative or positive tuberculin skin tests. Most (>98%) scored negative in an immunoblot test when the sera were analyzed at a 1:400 dilution. Approximately 40% of sera (diluted 1 in 400) from tuberculous patients (positive smears) recognized the antigen complex. The sensitivity of the test for patients suffering from extrapulmonary tuberculosis was similar to that for patients suffering from pulmonary tuberculosis but who had negative smears. The frequency of positive reactions among the patients suffering from other pulmonary diseases was similar to that among the control subjects. In tuberculous patients infected with human immunodeficiency virus, the sensitivity of the immunoblot test was significantly lower. Thus, this test based on an antigen complex used in an immunoblot assay to detect the presence of IgG antibody has a specificity of 98% and a sensitivity of 40%. The simultaneous use of different purified antigens, selected at the same high specificity level, may improve the sensitivity of such an assay.     

Serodiagnosis of tuberculosis and leprosy by enzyme immunoassay

Objective: To evaluate the use of serodiagnosis for tuberculosis and leprosy using mycobacterial antigen 38 kDa, with kits from Omega laboratories, to detect IgG by enzyme immunoassay (EIA).
Method: The study population consisted of 58 patients with evidence of tuberculous infection (culture of Mycobacterium tuberculosis complex or microscopic evidence), of whom 23 had pulmonary and 35 had extrapulmonary disease. There were six subjects who had recently been treated for tuberculosis, 11 patients on treatment for leprosy and 137 patients suspected of having tuberculosis on clinical or radiologic grounds (without laboratory evidence). A control group comprised 35 healthy individuals or patients suffering from diseases other than tuberculosis.
Results: The tests showed that there was a significant difference in antibody levels between the patients with active pulmonary disease, extrapulmonary tuberculosis and leprosy in comparison with the control group ( p <0.001). The sensitivities of the two tests together for proven pulmonary tuberculosis were 100% and 95.7% at 1.0–1.5 and >1.6 EIA cut-off points respectively, while the specificities were 88.5% and 100% at the same cut-off points. The sensitivities for extrapulmonary tuberculosis were 71.4% and only 51.4% at 1.0–1.5 and >1.6 EIA cut-off points. The test was positive in 30 (21.9%) of the 137 suspected patients, while 43 (31.4%) had an equivocal result and the remaining 64 (47.7%) suspects were definitely negative. There was again a significant difference in positivity rates between suspects and the control group.
Conclusions: Omega IgG test is useful in the serodiagnosis of active pulmonary tuberculosis and leprosy, but less sensitive in extrapulmonary disease, particularly in children. Equivocal results may only add to the evidence of tuberculosis in early or minimal disease.     

Serodiagnostic potentialities of enzyme-linked immunosorbent assay (ELISA) using mannophosphoinositides of Mycobacterium tuberculosis H37Rv

The serological response to mannophosphoinositides of Mycobacterium tuberculosis H₃₇Rv and to tuberculin-purified protein derivative (PPD) was examined by enzyme-linked immunosorbent assay (ELISA) in patients suffering from tuberculosis and related diseases. In sputum positive cases 94% samples were found to be positive to mannoside antigens and 77% to PPD, while in sputum negative cases, 71% of samples gave a positive reaction to mannosides and 54% to PPD. The high specificity of mannoside ELISA was demonstrated to be 97% in healthy individuals and 100% in patients suffering from other respiratory diseases, whereas PPD ELISA was 84% and 82% in healthy and infected patients respectively. Thus, ELISA is more specific and sensitive for mannosides than for PPD for the diagnosis of tuberculosis. However, antibodies to mannosides and PPD were detected in lepromatous as well as tuberculoid leprosy patients.     

Clinical evaluation of the Roche AMPLICOR PCR Mycobacterium tuberculosis test for detection of M. tuberculosis in respiratory specimens.

The reliability of the Roche AMPLICOR Mycobacterium tuberculosis test (AMPLICOR MTB) for the diagnosis of pulmonary tuberculosis was evaluated by testing 956 respiratory specimens from 502 patients and comparing results with results by culture and medical history. Of those 135 specimens that were culture positive for mycobacteria, 61 specimens from 31 patients grew M. tuberculosis. Fifty-two specimens were smear positive for acid-fast bacteria (AFB); M. tuberculosis was isolated from 41 of these specimens. On initial testing, the sensitivity and specificity of the AMPLICOR MTB assay, compared with culture, were 78.7 and 99.3%, respectively. After resolution of discrepancies (by review of medical history), the sensitivity, specificity, and positive and negative predictive values of the AMPLICOR MTB assay were 79.4, 99.6, 92.6, and 98.6%, respectively. Two specimens from two patients with no clinical evidence of tuberculosis were AMPLICOR MTB positive and culture positive for Mycobacterium avium complex. For AFB smear-positive specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 97.6, 100, 100, and 90.9%, respectively. For AFB smear-negative specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 40.0, 99.5, 69.2, and 98.7%, respectively. Our results support the use of AMPLICOR MTB for rapid diagnosis of tuberculosis in patients whose respiratory specimens are AFB smear positive. Further studies are needed to determine the most clinically relevant and cost-effective use of this assay with AFB smear-negative specimens.     

Cepheid GeneXpert MTB/RIF assay for Mycobacterium tuberculosis detection and rifampin resistance identification in patients with substantial clinical indications of tuberculosis and smear-negative microscopy results

The GeneXpert MTB/RIF assay was evaluated with microscopically negative and positive pulmonary and extrapulmonary specimens from patients with substantial clinical indications for tuberculosis. For the pulmonary samples, the sensitivity, specificity, and positive and negative predictive values were 90.6%, 94.3%, 93.5%, and 91.7%, and for the extrapulmonary samples, they were 100%, 91.6%, 50%, and 100%, respectively. For microscopically negative specimens, the respective values were 86.3%, 93%, 79%, and 95.6%. The assay correctly detected rifampin resistance in all but one specimen, which harbored a mixed population. The GeneXpert assay was highly effective for tuberculosis diagnosis and identification of rifampin-resistant strains in smear-negative samples.     

Use of multiepitope polyproteins in serodiagnosis of active tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Improved serodiagnosis of tuberculosis using two assay test.

An antigen capture immunoassay was developed for the detection of mycobacterial antigens in sera from patients with tuberculosis. The assay was evaluated together with an antibody measuring enzyme immunoassay in a clinical trial for serodiagnosis of tuberculosis. Sensitivity of the antibody assay for active pulmonary tuberculosis, including relapsed infections, was 75%, and specificity with other lung diseases was 97%. Sensitivity for extrapulmonary tuberculosis was 84.5% and specificity 84%. Sensitivity of the antigen assay for active tuberculosis was 45% with no false positive reactions. Combination of the results from the two assays increased total sensitivity to 96.5% with a positive predictive value of 0.81 and a negative value of 0.98. The two assay test was relatively simple to perform and offered improved serological diagnosis of tuberculosis over a single antibody test.     

Performance of Xpert MTB/RIF RUO assay and IS6110 real-time PCR for Mycobacterium tuberculosis detection in clinical samples

The Cepheid Xpert MTB/RIF research-use-only (RUO) assay and a laboratory-developed test (LDT) targeting IS6110 were evaluated and compared to mycobacterial culture as the gold standard. The performance characteristics of both molecular assays were determined by using 112 specimens from 90 patients, including 89 pulmonary specimens and 23 extrapulmonary specimens. Of the specimens tested, 37 (33%) were culture positive for the Mycobacterium tuberculosis complex; 29 were pulmonary, and 8 were extrapulmonary. Of these culture-positive specimens, 83% of the pulmonary specimens and 50% of the extrapulmonary specimens were smear positive. There was complete concordance between the smear-positive culture-positive specimens, independent of the anatomical site (100% sensitivity). The sensitivity of the MTB/RIF RUO assay for smear-negative specimens was 60% for pulmonary and 75% for extrapulmonary specimens, while the IS6110 LDT sensitivities were 40% and 0%, respectively. There was also complete concordance among the culture-negative specimens tested. Both assays showed 95% specificity, with four culture-negative specimens testing as positive. A review of patient records indicated that there was a high likelihood of the presence of M. tuberculosis complex DNA in the false-positive specimens. Biosafety analysis was performed and showed an acceptable reduction in organism viability using the processing methods described above. Both molecular assays are suitable for the detection of M. tuberculosis isolates in smear-positive pulmonary and extrapulmonary specimens, while the sensitivity of the detection of M. tuberculosis isolates in smear-negative specimens was variable.     

Diagnosis of Mycobacterium tuberculosis in cases of pulmonary infections from Jordanian hospitals

The aim of this study is to use the diagnostic utility of smear technique for the detection of Mycobacterium tuberculosis in clinical specimens obtained from patients suspected of having pulmonary tuberculosis. A total of 305 respiratory specimens (broncoalveolar lavage, sputum and pleural fluid) were collected from 298 patients having pulmonary infections as bronchitis, tuberculosis, pneumonia and other respiratory diseases. Results revealed that 25 specimens collected from 22 patients were positive (8.2%) by acid fast (AF) smear. Data indicated that the specificity and positive predictive value of the conventional smear assay were 100%, however, the sensitivity of the smear examination was 73.5%. Conventional smear technique actually detected M. tuberculosis in respiratory specimens and it could be applied for early and specific diagnosis of tuberculosis in such patients.     

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.     

Application of bleach method in diagnosis of extra-pulmonary tuberculosis

Extra pulmonary tuberculosis (TB) comprises 15% of the total tuberculosis cases. Bleach concentration method for demonstration of Acid fast bacilli (AFB) has been recently described for sputum. The aim of this study is to apply this method for demonstration of AFB in material obtained from extra-pulmonary sites and to correlate with cytology and conventional Ziehl Neelsen (Z N) staining. A total of 55 samples were studied from clinically suspected cases of extrapulmonary TB which included, FNA lymph nodes (17), abscesses drained from various body parts (18), body fluids (18) and skin scrapping (2). All the samples were processed for routine cytology, conventional ZN staining and bleach method followed by ZN staining. Out of 55 samples, 24(43.40%) were indicative of tuberculosis on cytology, 12(21.8%) were positive for AFB on conventional ZN staining, while the positivity increased to 39(70.90%) by Bleach method. Bleach solution is inexpensive and readily available in hospitals and its application has been proved in pulmonary tuberculosis. However to the best of our knowledge this is a pioneer study applied to the extra-pulmonary samples and the results of the present study shows improved detection of AFB.     

Interferon-γ release assays in the diagnosis of active tuberculosis disease in a low-incident setting: a 5-year review of data

The role of interferon-γ release assays in the diagnosis of active tuberculosis disease is uncertain, and recent guidelines do not support their routine use. We reviewed the clinical records of 415 patients who had a QuantiFERON-TB Gold In-Tube assay between 29 June 2005 and 28 October 2010 to determine its performance in the diagnosis of active tuberculosis disease in a low prevalence setting, specifically in human immunodeficiency virus (HIV) -positive and HIV-negative patients, those of UK and non-UK origin, and those with pulmonary and extrapulmonary disease. For the diagnosis of active tuberculosis disease the overall sensitivity of QuantiFERON-TB Gold In-Tube assay was 71.4% (95% CI 59.3–81.1), specificity was 81.0% (95% CI 75.5–85.6) and negative predictive value was 92.6% (95% CI 88.2–95.5). No significant difference in sensitivity was seen in culture-positive and culture-negative tuberculosis, in pulmonary and extrapulmonary disease, or with HIV infection. Specificity and negative predictive value were significantly higher in patients of UK origin compared with those of non-UK origin (89.3% (95% CI 83.3–93.3) and 97.1% (95% CI 92.7–98.9) versus 66.3% (95% CI 55.6–75.5) and 83.3% (95% CI 72.6–90.4)). Our study suggests that there may be a role for interferon-γ release assays in excluding active tuberculosis disease, particularly extrapulmonary disease, in patients originating from areas of low tuberculosis incidence, with a negative test highly predictive of a lack of active tuberculosis disease in this group. We cannot support the use of these assays in the diagnosis of active tuberculosis infection in patients from areas of higher incidence.     

Evaluation of the Characteristics of the Enzyme-Linked Immunospot Assay for Diagnosis of Active Tuberculosis in China

The purpose of this study was to evaluate the characteristics of the T-SPOT.TB test for the diagnosis of active tuberculosis (ATB) and to distinguish ATB from other diseases using a receiver operating characteristic (ROC) curve. A total of 535 patients with suspected active tuberculosis were enrolled in the study and divided into ATB and nonactive tuberculosis (NATB) groups, as well as pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) subgroups. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of the T-SPOT.TB test for the diagnosis of ATB were 84.95%, 85.12%, 82.94%, 86.93%, 5.71, and 0.18, respectively. The median number of spot-forming cells (SFCs) in the ATB group was higher than that in the NATB group (71 versus 1; P < 0.0001). The sensitivities in the PTB and EPTB subgroups were 92.31% and 81.77%. The areas under the curve (AUC) for the diagnosis of ATB using the T-SPOT.TB, early secreted antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP-10) were 0.906, 0.884, and 0.877, respectively. A cutoff of 42.5 SFCs for ATB yielded a positive predictive value of 100%. Our study shows that the T-SPOT.TB test is useful for the diagnosis of ATB. Utilizing an ROC curve to select an appropriate cutoff made it possible to discriminate ATB from NATB.     

Single-tube nested PCR in the diagnosis of tuberculosis.

AIMS: To evaluate the usefulness of a single-tube nested polymerase chain reaction (PCR) assay in the diagnosis of tuberculosis in 1497 pulmonary and 536 extrapulmonary specimens. METHODS: A single-tube nested PCR, utilising two sets of primers with different melting temperatures (88 degrees C for external primers; 70 degrees C for internal primers) to augment sensitivity and specificity without increasing the risk of amplicon contamination, was evaluated. Specimens were initially tested for the repetitive IS6110 sequences and if negative, retested for the universal 38 kilodalton sequence and for inhibitors. dUTP/Uracil-N-glycosylase and Instagene treatment were used to minimise contamination and the effect of inhibitors, respectively. RESULTS: Using culture as the gold standard, the overall sensitivity of the assay was 89% for pulmonary and 42% for extrapulmonary specimens. Sensitivity varied greatly with respect to sample type (92% for follow up specimens from a chest hospital and 70% for non-follow up specimens from a general hospital). The smear positivity rates were 15% for extrapulmonary specimens, and 69% and 45%, respectively, for follow up and non-follow up specimens from pulmonary sites. Specificity was 99.7%. Inhibitors were present more frequently in extrapulmonary than in pulmonary specimens (13.4% v 2.7%). CONCLUSION: Despite the high sensitivity of the PCR assay for the diagnosis of tuberculosis in pulmonary specimens, it was less effective in the extrapulmonary samples. This is probably because of the lower bacterial load in extrapulmonary specimens, the presence of more inhibitors adversely affecting the PCR assay and the higher volume of specimens used for culture.     

Use of Multiepitope Polyproteins in Serodiagnosis of Active Tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ~98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ~93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Diagnosis of extrapulmonary tuberculosis by smear, culture, and PCR using universal sample processing technology

Definitive and rapid diagnosis of extrapulmonary tuberculosis is challenging since conventional techniques have limitations. We have developed a universal sample processing (USP) technology for detecting mycobacteria in clinical specimens. In this study, this technology was evaluated blindly on 99 extrapulmonary specimens collected from 87 patients. USP-processed specimens were submitted to smear microscopy for detection of acid-fast bacilli (AFB), culture, and two PCR tests targeting devR (Rv3133c) and IS6110 gene sequences. On the basis of clinical characteristics, histology and cytology, conventional microbiology results, and response to antitubercular therapy, 68 patients were diagnosed with tuberculosis. Although USP smear and culture were significantly superior to conventional microbiology, which was not optimized (P < 0.0001), these approaches fell short of PCR tests (P < 0.0001). The low yields by smear and culture are attributed to the paucibacillary load in the specimens. The highest sensitivity in PCR was achieved when devR and IS6110 test results were combined; the sensitivity and specificity values were 83 and 93.8%, 87.5 and 100%, and 66.7 and 75%, respectively, in pleural fluid, needle-biopsied pleural tissue, and lymph node specimens. In conclusion, the application of USP technology, together with clinicopathological characteristics, promises to improve the accuracy and confidence of extrapulmonary tuberculosis diagnosis.     

Rapid diagnosis of extrapulmonary tuberculosis by PCR: impact of sample preparation and DNA extraction

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results.     

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.     

Xpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis: a diagnostic evaluation study

ObjectivesThe diagnosis of extrapulmonary tuberculosis (EPTB) is often made on clinical suspicion alone, resulting in both under- and overdiagnosis and relatively poor outcomes. In this study, we evaluated the clinical utility of the Xpert MTB/RIF on routinely collected extrapulmonary specimens in Ethiopia.MethodsThis study was carried out at Jimma University Specialized Hospital, Southwest Ethiopia. Extrapulmonary specimens were collected from 572 patients clinically suspected of suffering from EPTB. All specimens were tested for TB by smear microscopy, culture, and Xpert MTB/RIF. The diagnostic accuracy of Xpert MTB/RIF was calculated and compared to a composite reference standard (CRS), comprising clinical and laboratory results.ResultsIn total, 572 extrapulmonary specimens (279 lymph node, 159 pleural, 80 peritoneal, 45 cerebrospinal, and nine pericardial fluids) were tested. The pooled sensitivity and specificity of Xpert MTB/RIF were calculated to be 75% (95% CI 70–80) and 98% (95% CI 97–100) respectively when compared to the CRS. The highest sensitivity was documented for lymph node specimens (90%; 95% CI 86–94), moderate sensitivity for cerebrospinal fluid (53%; 95% CI 28–79), while the sensitivity was lowest for pleural (30%; 95% CI 17–44) and peritoneal (32%; 95% CI 12–51) fluids. Xpert MTB/RIF in addition detected rifampicin resistance in 13 patients, in perfect agreement with results from the line probe assay.ConclusionsXpert MTB/RIF may be used as initial diagnostic tool for testing of lymph node specimens from patients suspected of having TB lymphadenitis. The added value of Xpert MTB/RIF to diagnose pleural or peritoneal TB is limited by its poor sensitivity.     

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.