Tuberculosis Assay Research Articles

Diagnostic performance of multiplex cytokine and chemokine assay for tuberculosis

Simultaneous detection of multiple biomarkers might lead to improved diagnostic performance for Mycobacterium tuberculosis infection. In this study, we screened soluble biomarkers that had significant differences in patients with active tuberculosis and healthy controls and evaluated the diagnostic performance of the multiplex cytokine/chemokine assay. Overall, 178 patients with active pulmonary tuberculosis, 156 healthy individuals and 35 patients with bacterial pneumonia or lung cancer were evaluated. Among the 16 soluble biomarkers screened by the microbead-based multiplex assay, five cytokines/chemokines including IFN-γ, IP-10, MIG, TNF-α and IL-2 that showed most significant differences between active pulmonary tuberculosis patients and healthy controls were selected for further analysis. When analyzed individually, both IP-10 and MIG had sensitivity and specificity comparable to IFN-γ in detection of active TB. Combined detection of IFN-γ, IP-10 and MIG had significantly improved sensitivity and specificity as compared with individual cytokine and chemokine detection. The responsive levels of IFN-γ, IP-10, MIG, TNF-α and IL-2 were significantly lower in re-treatment pulmonary tuberculosis patients than in new tuberculosis patients. It is concluded that combined IFN-γ, IP-10, MIG multiplex detection had better diagnostic performance for tuberculosis than the individual cytokine/chemokine assays. The re-treatment pulmonary tuberculosis patients had poor responses to ESAT-6/CFP-10 peptides stimulation.

High Diagnostic Yield of Tuberculosis From Screening Urine Samples From HIV-Infected Patients With Advanced Immunodeficiency Using The Xpert MTB/RIF Assay

We determined the diagnostic yield of the Xpert MTB/RIF assay for tuberculosis (TB) when testing small volumes of urine from ambulatory HIV-infected patients before starting antiretroviral therapy in South Africa. Compared with a gold standard of sputum culture, the sensitivity of urine Xpert among those with CD4 cell counts of <50, 50-100, and >100 cells per microliter were 44.4%, 25.0%, and 2.7% (P = 0.001), respectively. Urine Xpert testing provides a means of rapid TB diagnosis in patients with advanced immunodeficiency and poor prognosis. These data are indicative of high rates of TB dissemination and renal involvement in this clinical population.

Positive predictive value of Interferon-gamma release assay for incident active tuberculosis in HIV-infected persons

Tuberculosis (TB) is the leading cause of death in HIV/AIDS. The incidence of active TB is 30 times higher in HIV-infected persons. Treatment of latent TB infection (LTBI) is pivotal to the control of TB. We aim to determine the positive predictive value of an interferon-gamma release assay, QuantiFERON-TB GOLD test (QFT), for incident active TB in HIV-infected persons.

Development of a diagnostic gene expression assay for tuberculosis and its use under field conditions in African buffaloes (Syncerus caffer)

The development of diagnostic tests for tuberculosis (TB) in exotic species is constrained by host biology and the limited availability of suitable assay reagents. As such, we evaluated a gene expression assay (GEA) which is easily modified for novel species and allows for initial sample processing under field conditions. African buffaloes (Syncerus caffer) were categorized using the single comparative intradermal tuberculin test, and blood from test-positive and test-negative animals was incubated for 20h in “Nil” tubes (containing saline) and “TB Antigen” tubes (containing Mycobacterium tuberculosis complex (MTC)-specific antigens) of a commercial human TB test, the QuantiFERON®-TB Gold (In-Tube) (QFT) assay. Blood samples were then stabilized in RNAlater® and transported to the laboratory for RNA extraction. A Custom TaqMan GEA was used to calculate the relative abundance of interferon-gamma (IFN-γ) mRNA in the TB Antigen tube compared to that in the Nil tube as a marker of immune activation in response to MTC antigen recognition. The GEA results from the two buffalo groups were compared and a cutoff value of 2.85 was calculated to differentiate between animals from these groups with a sensitivity of 80% (95% C.I.: 56–94%) and a specificity of 95% (95% C.I.: 75–100%). Further optimization of this assay could provide a highly useful tool for the diagnosis of MTC infection in exotic species.

A result analysis on phage amplified biologically assay and smear method for detecting Mycobacterium tuberculosis

Objective To compare the value of phage amplified biologically ( PhaB ) assay with that of smear method in detecting Mycobacterium tuberculosis.Methods The sputum specimens from 400 patients clinically diagnosed as pulmonary tuberculosis were detected by PhaB assay and smear method.The positive rate was counted.The sputum samples from 100 patients with nonpulmonary tuberculosis were detected by the same methods.The false positive rate was compared between the two groups.Results The positive rate of PhaB assay was significantly higher than that of smear method ( 58.5％ vs.29.5％,P＜ 0.05 )； and there was no significant difference between the two groups in false positive rate ( P ＞ 0.05 ).Conclusions Phage amplified biologically assay has a high positive rate and a low false positive rate as well as a shorter detection cycle.it can be used as a rapid diagnostic index in clinical application. Key words: Phage amplified biologically assay; Smear Method; Pulmonary Tuberculosis; Sputum specimens

Development of a Colorimetric Assay and Kinetic Analysis for Mycobacterium tuberculosis D-glucose-1-phosphate Thymidylyltransferase

dTDP-L-rhamnose as a sugar donor provides L-rhamnosyl residue in the synthesis of disaccharide linker (D-N-acetylglucosamine-L-rhamnose), the key structure of the Mycobacterium tuberculosis cell wall. Four enzymes are involved in the formation of dTDP-L-rhamnose and D-glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the first step of D-glucose-1-phosphate and dTTP to dTDP-D-glucose and PPi. The previous studies on RmlA essentiality proved RmlA as a potential target for antituberculosis drugs. However, there has not been a suitable assay for RmlA to screen inhibitors currently. In this study, the authors reported a microtiter plate–based colorimetric assay for RmlA enzyme activity. Using this assay, the kinetic properties of M. tuberculosis RmlA including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and kinetic parameters were determined. The establishment of the accurate and rapid colorimetric assay and kinetic analysis of M. tuberculosis RmlA will facilitate high-throughput screening of RmlA inhibitors.

Evaluation of Capilia TB assay for rapid identification of Mycobacterium tuberculosis complex in BACTEC MGIT 960 and BACTEC 9120 blood cultures

BackgroundCapilia TB is a simple immunochromatographic assay based on the detection of MPB64 antigen specifically secreted by the Mycobacterium tuberculosis complex (MTC). Capilia TB was evaluated for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 systems in Kampala, Uganda. Since most studies have mainly dealt with respiratory samples, the performance of Capilia TB on blood culture samples was also evaluated.MethodsOne thousand samples from pulmonary and disseminated tuberculosis (TB) suspects admitted to the JCRC clinic and the TB wards at Old Mulago hospital in Kampala, Uganda, were cultured in automated BACTEC MGIT 960 and BACTEC 9120 blood culture systems. BACTEC-positive samples were screened for purity by sub-culturing on blood agar plates. Two hundred and fifty three (253) samples with Acid fast bacilli (AFB, 174 BACTEC MGIT 960 and 79 BACTEC 9120 blood cultures) were analyzed for presence of MTC using Capilia TB and in-house PCR assays.ResultsThe overall Sensitivity, Specificity, Positive and Negative Predictive values, and Kappa statistic for Capilia TB assay for identification of MTC were 98.4%, 97.6%, 97.7%, 98.4% and 0.96, respectively. Initially, the performance of in-house PCR on BACTEC 9120 blood cultures was poor (Sensitivity, Specificity, PPV, NPV and Kappa statistic of 100%, 29.3%,7%, 100% and 0.04, respectively) but improved upon sub-culturing on solid medium (Middlebrook 7H10) to 100%, 95.6%, 98.2%, 100% and 0.98, respectively. In contrast, the Sensitivity and Specificity of Capilia TB assay was 98.4% and 97.9%, respectively, both with BACTEC blood cultures and Middlebrook 7H10 cultured samples, revealing that Capilia was better than in-house PCR for identification of MTC in blood cultures. Additionally, Capilia TB was cheaper than in-house PCR for individual samples ($2.03 vs. $12.59, respectively), and was easier to perform with a shorter turnaround time (20 min vs. 480 min, respectively).ConclusionCapilia TB assay is faster and cheaper than in-house PCR for rapid identification of MTC from BACTEC MGIT 960 and BACTEC 9120 culture systems in real-time testing of AFB positive cultures.

Monitoring of peptide-specific and gamma interferon-productive T cells in patients with active and convalescent tuberculosis using an enzyme-linked immunosorbent spot assay.

To establish a high-efficiency gamma interferon-specific enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT assay) for detection of tuberculosis (TB), peptides (E6, E7, and C14) and peptide mixtures (E6 plus E7 and E6 plus E7 plus C14) were used to monitor peripheral blood (PBL) samples from patients with pulmonary TB (PTB), as well as control samples. The positive detection rates of the five IFN-γ ELISPOT assays were 78.38%, 74.86%, 55.83%, 90.43%, and 91.51%, respectively, and there were similar detection rates between the two combined peptide mixture IFN-γ ELISPOT assays and the tuberculin skin test (TST) (90.62% versus 95.59%). No significant difference was found between the detection rates of the two combined peptide mixture IFN-γ ELISPOT assays and the T-SPOT.TB assay for 86 patients with PTB (P > 0.05), but the median number of spot-forming cells/10(6) cells (SFP value) for positive results was higher by the former than by the latter assay (P < 0.05). In contrast, the 29.93% positive detection rate and median SFP value of 482 by the two combined peptide mixture IFN-γ ELISPOT assays were significantly higher than the corresponding values of 14.29% and 152 by T-SPOT.TB assay for the same 147 community donors (P < 0.05). For nine PTB patients tracked, the SFP value of 7 for the two peptide mixture IFN-γ ELISPOT assays began to decrease from the second month after regular treatment. A relatively low, almost normal, SFP level was reached and maintained after the third or fourth month. Two in-house IFN-γ ELISPOT assays and the T-SPOT.TB assay could reduce the false-positive and false-negative detection rates of TST and sputum acid-fast staining. Therefore, these two combined peptide mixture IFN-γ ELISPOT assays have a potential advantage, beyond their greater specificity and sensitivity, for use in screening and detection of active TB infection (TBI) and latent TB infection (LTBI) in China.

Pathergy as a Cause of False-positive Tuberculin Skin Test

To the Editors: Differential diagnosis of intestinal tuberculosis (ITB) versus inflammatory bowel conditions can be challenging, especially in areas in which tuberculosis prevalence is very high or very low.1 Clinical presentation can be indistinguishable, and the extraintestinal manifestations of both conditions can be similar. Correct and prompt diagnosis is critical to avoid dire consequences, especially in the era of biologic therapies.2 The diagnosis of ITB can be very difficult, as both acid-fast bacilli staining and biopsy culture have low sensitivity, and the new polymerase chain reaction assays are not available in poor resource settings.1 Tuberculin skin test (TST) is often used in the diagnostic process. We report on a seldom considered cause of false-positive results to TST, possibly leading to a wrong diagnosis. A 15-year-old, Indian-born girl was admitted to our hospital because of a 6-month history of diarrhea and abdominal pain. She also presented recurrent painful oral ulcers, and ocular redness and pain in the left eye. One year earlier, she had received a diagnosis of tuberculosis-related erythema nodosum. A TST performed on that occasion resulted in extensive induration followed by blistering and ulceration of a 3-cm-wide skin area. Chest radiograph was negative for tuberculosis lesions, and she had been treated with a 6-month course of isoniazid. On admission to our hospital, laboratory tests showed elevated erythrocyte sedimentation rate, 78 mm/h, and fecal calprotectin (74 mg/kg of stools; normal values, <15), a marker of intestinal inflammation. Colonoscopy and video capsule endoscopy were performed to rule out ITB and inflammatory bowel disease. Sparse aphthous lesions were seen in the distal ileum. Polymerase chain reaction assay for Mycobacterium tuberculosis and staining for acid-fast bacilli were negative, as were also chest radiograph and interferon-gamma release assay results (QuantiFERON-TB Gold test; Cellestis Limited, Australia). An ophthalmology examination revealed acute anterior uveitis in the left eye. The patient's findings fulfilled the criteria for Behçet disease (BD) according to the International Study Group (recurrent oral ulceration, uveitis, erythema nodosum).3 Intestinal involvement in the form of ileal mucosal ulcers is also commonly reported in BD.4 She was treated with a 2-month course of oral prednisone and made a full recovery. The strongly positive TST can be explained as a pathergic reaction. Pathergy is characterized by the triggering of a cutaneous inflammatory response to minor trauma like needle prick. It is mainly described in BD and in some other neutrophilic dermatoses like pyoderma gangrenosum.5 Therefore, in presence of clinical and endoscopic finding compatible with both ITB and BD, the possibility of a false-positive TST caused by a pathergic reaction should be considered. On the other hand, BD and tuberculosis can coexist, so caution is mandatory. Serena Pastore, MD Samuele Naviglio, MD Alessandro Ventura, MD Institute for Maternal and Child Health IRCCS “Burlo Garofolo” University of Trieste Trieste, Italy

Mycobacterial Diseases of Animals 2012

Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, Mycobacterium ulcerans, and other mycobacteria are the etiologies of important diseases in humans and a wide range of animal species including cattle, sheep, goats, deer, possums, badgers, wild boar, elephants, dogs, cats, birds, amphibians, and fish. Moreover, M. bovis has a remarkably broad host range and is a serious zoonotic pathogen. In spite of centuries of investigation and costly eradication efforts, M. bovis remains an important pathogen at the interface of humans, livestock, and wildlife. M. avium subsp. paratuberculosis is one of the most widespread pathogens of dairy cattle, causing decreased production, protracted diarrhea, and emaciation. This 2nd special issue on mycobacterial diseases of animals contains 16 papers comprising 4 reviews and 12 original research papers on various topics including immunology, epidemiology, microbiology, genomics, vaccinology, and pathology. Authors from 9 different countries provide a diverse examination of mixed topics including M. tuberculosis, M. bovis, and M. avium subsp. paratuberculosis. The issue begins with G. M. Kassa et al. examining a cross-section of Ethiopian small ruminants and describing the isolation of M. tuberculosis from goats and stressing important zoonotic implications. From Spain, B. Beltran-Beck and colleagues review the significant progress on oral vaccination of Eurasian wild boar with the vaccine M. bovis BCG and a new heat-killed M. bovis vaccine. R. A. Skuce et al. review important risk factors for bovine TB herd breakdowns in the United Kingdom and Ireland. The next 3 papers focus on immunology and diagnostics beginning with B. Fernandez et al. and their analysis of an ELISA for bovine paratuberculosis. J. T. Nelson and colleagues evaluate a rapid serologic test for tuberculosis diagnosis in several deer species, while M. L. Mon et al. search for improved antigens for use in bovine paratuberculosis diagnosis. M. V. Palmer and collaborators review in detail wildlife reservoirs of M. bovis and the potential for disease transmission at the livestock-wildlife-human interface. C. Mackintosh and colleagues from New Zealand describe experimental infection of red deer with M. avium subsp. paratuberculosis and examine the clinical, immunological, and pathological outcomes. Rapid diagnostic tests for both bovine tuberculosis and paratuberculosis are high priorities in the veterinary medical field, and A. Wadhwa et al. review current and future platforms for diagnostic assays. A. Lim and colleagues at Michigan State University use genomics to examine differential gene expression between true M. bovis-positive and -false-positive cattle. Mycobacterium bovis can infect numerous species, but not all species have maintenance host potential. Intraspecies transmission is requisite to maintenance host potential. Fenton et al. examine potential intraspecies transmission among Virginia opossums in an experimental aerosol challenge model. Possible species differences in susceptibility to M. bovis infection lead G. Ameni et al. to explore potential differences in immune responses to M. bovis between Bos indicus and Bos taurus cattle in Ethiopia. C. Furphy and colleagues from Ireland use the molecular epidemiological tools of spoligotyping and VNTR to examine strain types of M. bovis in badgers and show that individual badgers can be infected with multiple strains of M. bovis. One useful serological assay for tuberculosis in cattle is the multiantigen print immunoassay, which S. D. Fitzgerald et al. examine, using various interpretation criteria and suggest its use as a possible supplemental test to intradermal tuberculin testing. K. P. Lyashchenko and colleagues describe the uncommon infection of a horse with M. tuberculosis and discuss possible zoonotic concerns. The highlands of Cameroon are the setting for J. Awah-Ndukum and collaborators as they use traditional tuberculin skin testing and novel serological tests to determine the prevalence of bovine tuberculosis and explore possible modifications of test interpretation to improve disease surveillance. The editors thank the many authors for their efforts in the experimentation, labor, and time reflected in each manuscript. The lead editor thanks all editors for time spent reviewing, assigning reviews, and commenting on the many manuscripts submitted. It is the hope of the editors that this 2012 issue will prove useful to investigators, policy makers, and veterinarians involved in the study of mycobacterial diseases of animals. Mitchell V. Palmer Michael D. Welsh Jesse M. Hostetter

Clinical Utility of Two Interferon-gamma Release Assays on Pleural Fluid for the Diagnosis of Tuberculous Pleurisy

BackgroundThe release of interferon-gamma (IFN-γ) by T lymphocytes increases after rechallenge with Mycobacterium tuberculosis antigen, especially, at a localized site of tuberculosis (TB) infection. We aimed to compare the clincial efficacy of two commercial IFN-γ release assays from pleural fluid for the diagnosis in tuberculous pleurisy.MethodsWe performed T-SPOT.TB and QuantiFERON-TB Gold tests simultaneously on pleural fluid and peripheral blood samples from patients with pleural effusion, in South Korea, an area with intermediate TB burden.ResultsThirty-six patients were enrolled prospectively, and tuberculous pleurisy was found in 21 patients. Both the numbers of IFN-γ secreting T cells and the concentration of IFN-γ were greater in the pleural tuberculous group, comparing with the non-tuberculous group. Moreover, in the tuberculous group, there was a significant difference in IFN-γ producing spot-forming cells using the T-SPOT.TB method between pleural fluid and peripheral blood. The receiver operating characteristic (ROC) curve, was the greatest for pleural fluid T-SPOT.TB test, followed by peripheral blood T-SPOT.TB test, peripheral blood QuantiFERON-TB Gold test, and pleural fluid QuantiFERON-TB Gold test (area under the ROC curve of 0.956, 0.890, 0.743, and 0.721, respectively). The T-SPOT.TB assay produced less indeterminate results than did QuantiFERON-TB Gold assay in both pleural fluid and peripheral blood.ConclusionThese findings suggest that the pleural fluid T-SPOT.TB test could be the most useful test among the IFN-γ release assays for diagnosing tuberculous pleurisy in an area with an intermediate prevalence of TB infection.

Point-of-Care Urine Antigen Screening Tests for Tuberculosis and Cryptococcosis: Potential for Mortality Reduction in Antiretroviral Treatment Programs in Africa

TO THE EDITOR—Between 8% and 26% of patients enrolling in antiretroviral treatment (ART) services in sub-Saharan Africa die within the first year of therapy, and a majority of these deaths occur within the first few months [1]. Mortality risk is particularly high among those with the lowest baseline CD4 cell counts, and tuberculosis and cryptococcal meningitis are leading causes [1]. Packages of interventions to reduce mortality need to include specific measures to target these 2 diseases [2]. Previous studies have highlighted that much of the burden of undiagnosed prevalent cryptococcosis and tuberculosis among patients enrolling in ART services can be diagnosed by careful systematic screening of patients at baseline by testing serum samples for cryptococcal antigen and culturing sputum samples for Mycobacterium tuberculosis [3, 4]. However, screening using these methods requires laboratory-based processing of clinical samples remote from the site of care. Moreover, mycobacterial culture is slow. Thus, delays in availability of test results greatly undermine the potential for screening to rapidly direct appropriate clinical management and thereby improve prognosis. Simple, rapid, and low-cost point-of-care assays are needed. We therefore read with great interest the findings of Jarvis and colleagues published in Clinical Infectious Diseases regarding the evaluation of a novel lateral-flow assay for cryptococcal antigen [5]. It was shown that this point-of-care screening test could be applied not only to serum and plasma, but also to urine samples, providing results within 10 minutes. Positive results might be used to direct further investigation or preemptive antifungal therapy. This publication coincided with our evaluation of a low-cost point-of-care assay for tuberculosis [6]; this detects the mycobacterial antigen lipoarabinomannan (LAM), which is present in the urine of a proportion of patients with human immunodeficiency virus (HIV)–associated tuberculosis. This simple, commercially available strip test (Determine TB-LAM Ag; Alere) had a diagnostic accuracy that was similar to that of the laboratory-based enzyme-linked immunosorbent assay version of the assay [7]. It provided a highly specific tuberculosis diagnosis in two-thirds of patients who had culture-confirmed tuberculosis and very low CD4 cell counts [6]. Similar to the cryptococcal antigen screening tool, the urine LAM test is a simple lateral-flow assay that requires no sample preparation, laboratory hardware, or power supply and provides results in just 25 minutes. The development of these point-of-care diagnostic screening tests for cryptococcosis and tuberculosis potentially represents a major step forward for the clinical management of HIV-infected patients entering ART programs or requiring hospital admission in resource-limited settings. The assays might be used to systematically screen all new patients, regardless of symptoms, who have low CD4 cell counts (eg, <100 or <150 cells/μL). This patient subgroup has a high prevalence of both diseases, and rapid diagnosis and initiation of appropriate management are needed in view of high mortality risk. Simple urine antigen tests for other key pathologies, such as pneumococcal pneumonia [8], might also be useful in these clinical settings and should be evaluated. After development of these point-of-care assays, studies are now needed to determine how to best incorporate these new tools into clinical management and to assess their impact on patient outcomes.

The Use of Selected Commercial Molecular Assays for the Microbiological Diagnosis of Tuberculosis

Introduction: We performed a retrospective assessment of the AMPLICOR Mycobacterium tuberculosis (MTB) assay for the molecular diagnosis of tuberculosis based on our own determinations between 1999 and 2009 and a preliminary assessment of the Xpert MTB/RIF system, which we are currently using. Materials and methods: The study groups comprised 1875 samples (including 104 inhibited samples) and 213 samples, respectively. Results: The sensitivities of the AMPLICOR MTB and the Xpert MTB/RIF assays were 81.9% and 81.8%, respectively, and their specificities were 97.2% and 99.5%, respectively, versus culture on Loewenstein-Jensen medium. Both assays showed a considerable difference in sensitivity depending on whether the test samples were smear-positive (AFB+) or smearnegative (AFB–). The sensitivities of the AMPLICOR MTB and the Xpert MTB/RIF assays were 97.8% and 100.0%, respectively, for AFB+ samples and 58.1% and 50.0%, respectively, for AFB– samples. Conclusions: Our results confirm full usefulness of the Xpert MTB/RIF assay for routine diagnosis in the case of smearpositive clinical samples.

Tuberculosis-specific T-cell response after recent treatment and remote cure

To the Editors: Tuberculosis (TB) remains a major global health problem. A major factor contributing to the current TB epidemic is the resistance of Mycobaterium tuberculosis to first-line anti-TB treatment. Therefore, there is a need for an appropriate and reliable tool to evaluate treatment efficacy. Several studies using the T-SPOT®. TB assay (Oxford Immunotec Ltd, Oxford, UK) reported a decline in the number of spots after successful anti-TB treatment, particularly in the 6-kDa early secretory antigenic target (ESAT-6) panel [1–3]; however, to our knowledge, no data have been published on cured TB patients who contracted TB before isoniazid and rifampicin became available. Therefore, the naturally long-term range of ESAT-6- and culture filtrate protein (CFP)-10-specific T-cell responses in patients with TB who did not receive modern anti-TB treatment is not known. Because most patients in prior studies were born in high-burden, TB-endemic countries, the T-cell kinetics might be influenced by reinfection. Therefore, the aim of this report is to compare the ESAT-6- and CFP-10-specific T-cell responses in a low-burden country with a low risk of reinfection between patients with a history of active TB before 1955, and patients with recently treated active TB. Venous blood for T-SPOT®. TB was collected from 24 outpatient subjects with a history of active TB before 1955 (group 1), obtained by clinical history-taking, patients’ medical records or documentation of a sanatorium admission. 16 of these patients had been admitted to a sanatorium, two patients underwent surgery and six patients were treated at home with para-aminosalicylic acid therapy or bedrest. Furthermore, 27 patients with recent active TB (group 2) were prospectively enrolled between March and May 2008. Active TB was defined as fulfilling one or more of the following criteria: 1) positive culture for M. tuberculosis (n=22); 2) positive PCR for M. …

Diagnostic accuracy of a low-cost, urine antigen, point-of-care screening assay for HIV-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study

SummaryBackgroundThe diagnostic accuracy of sputum smear microscopy and routine chest radiology for HIV-associated tuberculosis is poor, and culture-based diagnosis is slow, expensive, and is unavailable in most resource-limited settings. We assessed the diagnostic accuracy of a urine antigen test Determine TB-LAM Ag (Determine TB-LAM; Alere, Waltham, MA, USA) for screening for HIV-associated pulmonary tuberculosis before antiretroviral therapy (ART).MethodsIn this descriptive study, consecutive adults referred to a community-based ART clinic in Gugulethu township, South Africa, were all screened for tuberculosis by obtaining sputum samples for fluorescence microscopy, automated liquid culture (gold-standard test), and Xpert MTB/RIF assays (Cepheid, Sunnyvale, CA, USA) and urine samples for the Clearview TB-ELISA (TB-ELISA; Alere, Waltham, MA, USA) and Determine TB-LAM test. Patients with Mycobacterium tuberculosis cultured from one or more sputum samples were defined as cases of tuberculosis. The diagnostic accuracy of Determine TB-LAM used alone or combined with sputum smear microscopy was compared with that of sputum culture and the Xpert MTB/RIF assay for all patients and subgroups of patients stratified by CD4 cell count.FindingsPatients were recruited between March 12, 2010, and April 20, 2011. Of 602 patients enrolled, 542 were able to provide one or more sputum samples, and 94 had culture-positive tuberculosis (prevalence 17·4%, 95% CI 14·2–20·8). Complete results from all tests were available for 516 patients (median CD4 count, 169·5 cells per μL; IQR 100–233), including 85 culture-positive tuberculosis, 24 of whom (28·2%, 95% CI 19·0–39·0) had sputum smear-positive disease. Determine TB-LAM test strips provided results within 30 min. Agreement was very high between two independent readers of the test strips (κ=0·97) and between the test strips and TB-ELISA (κ=0·84). Determine TB-LAM had highest sensitivity at low CD4 cell counts: 66·7% (95% CI 41·0–86·7) at <50 cells per μL, 51·7% (32·5–70·6) at <100 cells per μL, and 39·0% (26·5–52·6) at <200 cells per μL; specificity was greater than 98% for all strata. When combined with smear microscopy (either test positive), sensitivity was 72·2% (95% CI 46·5–90·3) at CD4 counts less than 50 cells per μL, 65·5% (45·7–82·1) at less than 100 cells per μL, and 52·5% (39·1–65·7) at less than 200 cells per μL, which did not differ statistically from the sensitivities obtained by testing a single sputum sample with the Xpert MTB/RIF assay.InterpretationDetermine TB-LAM is a simple, low-cost, alternative to existing diagnostic assays for tuberculosis screening in HIV-infected patients with very low CD4 cell counts and provides important incremental yield when combined with sputum smear microscopy.FundingWellcome Trust.

Corticosteroids and immunosuppressive therapy influence the result of QuantiFERON TB Gold testing in inflammatory bowel disease patients

IntroductionLatent tuberculosis infection is detected by the tuberculin skin test before treating with anti-Tumour-Necrosis factor alpha (anti TNFα) reagents. More accurate are Interferon gamma release assays (IFNγ release assays) to identify patients with latent tuberculosis. Because of a positive control in this assay, it is possible to identify those patients in which a result of tuberculosis testing is not available due to a lack of stimulation capacity of lymphocytes (indeterminate result). Patients suffering from IBD are often treated with immunosuppressive agents, which may influence the results of tuberculosis testing. AimThe aim is to investigate the influence of immunosuppressive agents on the outcome of IFNγ-release assay. Methods50 consecutive patients were documented before introducing anti-TNF-treatment in this single centre study between April 2009 and April 2010. Data of INFγ release assay for latent tuberculosis, skin test and laboratory data and current medication were enrolled. ResultsFor the period of one year data of 45 consecutive patients was available for statistical analysis. 24 patients out of 45 (corresponding to 53.3%) received at least low doses of corticoid treatment and 27 patients out of 45 (corresponding to 60.0%) received immunosuppressive agents. 13 patients out of 45 (corresponding to 28.9%) had an indeterminate result of the QuantiFERON test. A correlation between the indeterminate result and combination therapy of corticosteroids was found. The concomitant therapy of immunosuppressive agents lead to a lower IFN release but no significance was found. ConclusionsSteroid treatment and further combination therapy with immunosuppressive agents lead to a high risk of indeterminate QuantiFERON test.

Polymeric Sequence Probe for Single DNA Detection

A polynucleotide probe, polymeric sequence probe (PSP), was developed for single molecular detections. PSP is a single-stranded DNA molecule with ~2000 tandem repeat target-binding sequences and label-binding sequences. A single PSP can bind to multiple fluorescent complementary oligos to generate a strong fluorescence signal. Single target molecules bound to PSPs can be clearly visualized by a conventional fluorescence microscope. An ultrasensitive PSP-based assay for Mycobacterium tuberculosis was demonstrated.

Analysis of Factors Lowering Sensitivity of Interferon-γ Release Assay for Tuberculosis

BackgroundImperfect sensitivity of interferon-γ release assay (IGRA) is a potential problem to detect tuberculosis. We made a thorough investigation of the factors that can lead to false negativity of IGRA.MethodsWe recruited 543 patients with new smear-positive pulmonary tuberculosis in Hanoi, Viet Nam. At diagnosis, peripheral blood was collected and IGRA (QuantiFERON-TB Gold In-Tube) was performed. Clinical and epidemiological information of the host and pathogen was collected. The test sensitivity was calculated and factors negatively influencing IGRA results were evaluated using a logistic regression model in 504 patients with culture-confirmed pulmonary tuberculosis.ResultsThe overall sensitivity of IGRA was 92.3% (95% CI, 89.6%–94.4%). The proportions of IGRA-negative and -indeterminate results were 4.8% (95% CI, 3.1%–7.0%) and 3.0% (95% CI, 1.7%–4.9%). Age increased by year, body mass index <16.0, HIV co-infection and the increased number of HLA-DRB1*0701 allele that patients bear showed significant associations with IGRA negativity (OR = 1.04 [95% CI, 1.01–1.07], 5.42 [1.48–19.79], 6.38 [1.78–22.92] and 5.09 [2.31–11.22], respectively). HIV co-infection and the same HLA allele were also associated with indeterminate results (OR = 99.59 [95% CI, 15.58–625.61] and 4.25 [1.27–14.16]).ConclusionsAging, emaciation, HIV co-infection and HLA genotype affected IGRA results. Assessment of these factors might contribute to a better understanding of the assay.

Diagnostic performance of whole-blood interferon-γ assay and enzyme-linked immunospot assay for active tuberculosis,

The aim of this study was to compare the diagnostic performance of 2 interferon-γ release assays, an enzyme-linked immunospot assay (T-SPOT.TB; Oxford Immunotec Ltd., Oxford, UK) and the QuantiFERON-TB Gold in-Tube assay (QFT-GIT; Cellestis Ltd., Carnegie, Australia), in patients with suspected active tuberculosis (TB). From October 2009 to October 2011, a total of 200 patients with suspected TB were enrolled. Clinical and microbiological characteristics of the patients were collected and blood samples were obtained for T-SPOT.TB and QFT-GIT assays. Among the 200 subjects, 98 (49%) had culture-confirmed TB, 18 (9%) had probable TB, and the remaining 84 (42%) subjects did not have TB. The sensitivity, specificity, positive predictive value, and negative predictive value for active TB diagnosis by the T SPOT. TB were 83%, 71%, 81%, and 75%, respectively. For QFT-GIT, the sensitivity, specificity, positive predictive value, and negative predictive value for active TB diagnosis were 66%, 76%, 80%, and 62%, respectively. The QFT-GIT assay resulted in more indeterminate and false-negative results than the T-SPOT.TB assay, especially in immunocompromised patients. In conclusion, T-SPOT.TB had a higher sensitivity and resulted in fewer indeterminate results than the QFT-GIT assay for diagnosing active TB.

Screening for HIV-Associated Tuberculosis and Rifampicin Resistance before Antiretroviral Therapy Using the Xpert MTB/RIF Assay: A Prospective Study

The World Health Organization has endorsed the Xpert MTB/RIF assay for investigation of patients suspected of having tuberculosis (TB). However, its utility for routine TB screening and detection of rifampicin resistance among HIV-infected patients with advanced immunodeficiency enrolling in antiretroviral therapy (ART) services is unknown. Consecutive adult HIV-infected patients with no current TB diagnosis enrolling in an ART clinic in a South African township were recruited regardless of symptoms. They were clinically characterised and invited to provide two sputum samples at a single visit. The accuracy of the Xpert MTB/RIF assay for diagnosing TB and drug resistance was assessed in comparison with other tests, including fluorescence smear microscopy and automated liquid culture (gold standard) and drug susceptibility testing. Of 515 patients enrolled, 468 patients (median CD4 cell count, 171 cells/µl; interquartile range, 102-236) produced at least one sputum sample, yielding complete sets of results from 839 samples. Mycobacterium tuberculosis was cultured from 81 patients (TB prevalence, 17.3%). The overall sensitivity of the Xpert MTB/RIF assay for culture-positive TB was 73.3% (specificity, 99.2%) compared to 28.0% (specificity, 100%) using smear microscopy. All smear-positive, culture-positive disease was detected by Xpert MTB/RIF from a single sample (sensitivity, 100%), whereas the sensitivity for smear-negative, culture-positive TB was 43.4% from one sputum sample and 62.3% from two samples. Xpert correctly identified rifampicin resistance in all four cases of multidrug-resistant TB but incorrectly identified resistance in three other patients whose disease was confirmed to be drug sensitive by gene sequencing (specificity, 94.1%; positive predictive value, 57%). In this population of individuals at high risk of TB, intensive screening using the Xpert MTB/RIF assay increased case detection by 45% compared with smear microscopy, strongly supporting replacement of microscopy for this indication. However, despite the ability of the assay to rapidly detect rifampicin-resistant disease, the specificity for drug-resistant TB was sub-optimal.