Giulia Lombardi

Clinical validation of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis

Extrapulmonary tuberculosis (EPTB) accounts for more than 20% of tuberculosis (TB) cases. Xpert MTB/RIF (Xpert) (Cepheid, Sunnyvale, CA, USA) is a fully automated amplification system, for which excellent results in the diagnosis of pulmonary TB in highly endemic countries have been recently reported. We aimed to assess the performance of the Xpert system in diagnosing EPTB in a low incidence setting. We investigated with Xpert a large number of consecutive extrapulmonary clinical specimens (1,476, corresponding to 1,068 patients) including both paediatric (494) and adult samples. We found, in comparison with a reference standard consisting of combination of culture and clinical diagnosis of TB, an overall sensitivity and specificity of 81.3% and 99.8% for Xpert, while the sensitivity of microscopy was 48%. For biopsies, urines, pus and cerebrospinal fluids the sensitivity exceeded 85%, while it was slightly under 80% for gastric aspirates. It was, in contrast, lower than 50% for cavitary fluids. High sensitivity and specificity (86.9% and 99.7%, respectively) were also obtained for paediatric specimens. Although the role of culture remains central in the microbiological diagnosis of EPTB, the sensitivity of Xpert in rapidly diagnosing the disease makes it a much better choice compared to smear microscopy. The ability to rule out the disease still remains suboptimal.

Immunogenetic basis of environmental lung disease: lessons from the berylliosis model

The role of genetic factors has been hypothesized in the pathogenesis of a number of chronic inflammatory lung diseases. The genes of the major histocompatibility complex (MHC) locus on human chromosome 6 have been identified as important determinants in diseases caused both by inorganic and organic compounds such as beryllium, gold, acid anhydrides, isocyanates and grass pollens. Since many environmental factors are the determinants of the immunopathogenesis of asthma, pulmonary granulomatous disorders, hypersensitivity pneumonitis and fibrotic lung disorders, an understanding of the interaction between environmental factors is crucial to epidemiology, prevention and treatment of these disorders. Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of beryllium dusts. A human leukocyte antigen class II marker (HLA-DP Glu69) has been found to be strongly associated with the disease. In in vitro studies, the gene has been shown to play a direct role in the immunopathogenesis of the disease. In human studies, the gene has been shown to confer increased susceptibility to beryllium in exposed workers, thus suggesting that HLA gene markers may be used as epidemiological probes to identify population groups at higher risk of environmental lung diseases, to identify environmental levels of lung immunotoxicants that would be safe for the entire population and to prevent disease risk associated with occupation, manufactured products and the environment. Studies on the associations between human leukocyte antigens and chronic inflammatory lung disorders are reviewed in the context of the berylliosis model.

First independent evaluation of QuantiFERON-TB Plus performance

Tuberculosis elimination requires an effective strategy to diagnose and treat people infected with Mycobacterium tuberculosis who would otherwise be at high risk of developing and transmitting active disease [1, 2]. The diagnostic tools for latent tuberculosis infection (LTBI) are the tuberculin skin test (TST) and the T-cell interferon-γ release assays (IGRAs). Two IGRAs are commercially available, QuantiFERON-TB Gold In-Tube (QFT-GIT) (Qiagen, Hilden, Germany) and T-SPOT.TB (Oxford Immunotec, Abingdon, UK). Compared to the TST, IGRAs offer operational advantages and higher specificity in the bacille Calmette–Guérin (BCG)-vaccinated population [3], and they are at least as sensitive for LTBI [4]. However, IGRAs have limitations: reduced sensitivity in children and immunocompromised subjects, including HIV-infected individuals [3, 4]; failure to discriminate between active tuberculosis and LTBI; and poor correlation with the risk of progression to active disease [3]. QuantiFERON-TB Plus improves sensitivity for active TB and maintains high specificity among unvaccinated controls http://ow.ly/XjYPK

Diagnosis of smear-negative tuberculosis is greatly improved by Xpert MTB/RIF

Background Diagnosis of pulmonary (PTB) and extra-pulmonary tuberculosis (EPTB) in smear-negative patients can be difficult. We assessed retrospectively the performance of Xpert MTB/RIF system (Xpert, Cepheid) in diagnosing smear-negative tuberculosis (TB), which represents the most common form of TB in a low incidence setting. Methods Performance of Xpert was compared to acid-fast microscopic examination using Ziehl-Neelsen (ZN) stain in patients with culture-confirmed TB. Results 386 Mycobacterium tuberculosis (MTB) culture-positive samples were detected out of 5170 specimens tested with smear microscopy, Xpert and culture: 323 were both culture- and Xpert-positive, and 63 culture-positive only. Of these, 234 (60.6%) were smear-negative. In addition Xpert detected 40 probable TB cases, based on clinical findings, which were culture-negative. Compared to culture, Xpert showed an overall sensitivity of 83.7% and a specificity of 99.1%; sensitivity was higher for respiratory samples (86.5%) than for non-respiratory samples (76.8%). Xpert sensitivity for smear-negative culture-confirmed TB was 73.1% and was not influenced by TB localization. As sensitivity of microscopy alone was poor (39.4%), Xpert improved both diagnosis of pulmonary TB (Δ = 36.5%) and extra-pulmonary TB (Δ = 63.4%). Conclusions Xpert MTB/RIF is a sensitive method for rapid diagnosis of TB compared to the conventional ZN staining. Xpert can serve as a sensitive and time-saving diagnostic method for microbiological diagnosis of smear-negative TB in countries with a low TB prevalence.

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

ABSTRACT 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.

Quantiferon-tb Gold In-tube Improves Tuberculosis Diagnosis in Children

Background: The diagnostic accuracy of Quantiferon-TB Gold In-Tube (QFT-IT) is uncertain in the pediatric population, while tuberculin skin test (TST) is still conventionally used despite its limitations. The aim of this study was to compare the performance of QFT-IT with TST in a large cohort of children screened for tuberculosis (TB) infection because of contact tracing, suspected TB, arrival from endemic country or immunosuppressive therapy. Methods: A retrospective analysis was conducted on 517 children 0–14 years of age evaluated at the pediatric unit of the S. Orsola-Malpighi University Hospital of Bologna, Italy; 366 of them were also tested with TST. Results were analyzed for Calmette-Guérin bacillus vaccination, country of origin, reason for testing, diagnosis and age. Results: The overall agreement between the 2 tests was 89.9%, but it was highly affected by Calmette-Guérin bacillus vaccination (P < .0001). According to diagnosis and age, QFT-IT detected latent tuberculous infection cases better than TST in all age groups. Sensitivity for diagnosing active TB in symptomatic children was higher for QFT-IT than TST (93.3% vs. 86.5%), especially in children younger than 2 years, while specificity was high for both tests (99.3% and 98.8%, respectively). Low rate of indeterminate QFT-IT results (3.9%) was not differently distributed among age groups, but was associated with diagnosis of TB exclusion (P < 0.0001), mainly pneumonia (35%), and to Italian children (P = 0.0024). Conclusions: Despite the concern about the use of QFT-IT in children because of their immature immune system, our results suggest the preferential use of QFT-IT as a support tool for diagnosis and management of TB, even in infants.

Five-year surveillance of human tuberculosis caused by Mycobacterium bovis in Bologna, Italy: an underestimated problem.

Human tuberculosis (TB) caused by Mycobacterium bovis surveillance is affected by a lack of data. The aims of the present study were: (i) to estimate the proportion of human TB caused by M. bovis over a period of 5 years in Bologna, Northern Italy, which, like most Western European countries, has been declared bovine TB-free; (ii) to compare the genetic profiles of M. bovis strains identified in humans with those circulating in cattle in the last 15 years in Italy. Among 511 TB patients, the proportion of human TB caused by M. bovis was 1·76%, significantly associated to extra-pulmonary localization (P = 0·004) and to being elderly (P < 0·001) and Italy-born (P = 0·036). The molecular epidemiology analysis by spoligotyping and Multilocus Variable Tandem Repeat Analysis confirmed that most M. bovis strains from Italy-born patients matched those circulating in cattle herds in Italy between 2001 and 2016. Two cases of Mycobacterium bovis BCG infection were also characterized. In conclusion, the rate of human TB caused by M. bovis was not negligible, highlighting the relevance of molecular typing in evaluating the effectiveness of programmes designed to eradicate TB in cattle in Italy.

Improvement of Mycobacterium tuberculosis detection by Xpert MTB/RIF Ultra: A head-to-head comparison on Xpert-negative samples

Background The new Xpert MTB/RIF Ultra assay (Ultra, Cepheid, Sunnyvale, USA) is a cartridge-based automated diagnostic test that can simultaneously identify Mycobacterium tuberculosis complex (MTB) and resistance to Rifampicin (RIF). With respect to the previous version Xpert MTB/RIF assay (Xpert), IS6110/IS1081 repetitive elements probes have been added allowing the detection of lower MTB load, defined by the new semi-quantitative category “trace” with indeterminate RIF resistance. The aim of this study was to evaluate performance of the new version Ultra on Xpert-negative, but TB culture-positive clinical samples. Methods The de-identified frozen samples (-20 °C) collected over a 4-year period (February 2014-October 2017), which had previously resulted smear-negative, Xpert-negative but MTB culture-positive, were analyzed with Ultra. The de-frosted samples were loaded into the cartridge using the same process as the previous version, according to manufacturer’s instruction. Results During the study period 382 MTB culture-positive samples were archived: 314 resulted Xpert-positive and 68 Xpert-negative. Thirty-one of the 68 Xpert-negative samples resulted positive with Ultra, with an overall improvement in MTB detection of 45.6%. Out of 36 Xpert-negative respiratory samples, 18 resulted Ultra-positive with the following semi-quantitative loads: “low”(n = 1), “very low”(n = 11), “trace”(n = 6), with an improvement in MTB detection of 50%. The best performance was achieved on bronchoalveolar lavage specimens (53.8%). Out of 32 Xpert-negative non-respiratory samples, 13 resulted Ultra-positive with the following semi-quantitative loads: “very low”(n = 7), “trace”(n = 6), with an improvement in MTB detection of 40.6%. The best performance was achieved on biopsies (55.6%) and lymph nodes (50%). The new category “trace” detected 12 out of the 31 Ultra-positive MTB samples; in the remaining 19 samples RIF susceptibility was determined with 100% concordance with the phenotypic susceptibility test. The mean time to positivity of samples found negative by Ultra was significantly longer in comparison to positive samples in liquid culture. Conclusions Our results are consistent with the few studies published so far and confirm the better performance of Ultra compared to the previous version in both respiratory and non-respiratory smear-negative samples, with an overall improvement of 45.6%.

Does biological therapy affect interferon-γ release assay response? A long-term follow-up of patients with psoriasis using QuantiFERON-TB

DEAR EDITOR, Patients with psoriasis treated with biological drugs are at increased risk of reactivation of latent tuberculosis infection (LTBI). Therefore screening for active or latent TB is mandatory before initiating therapy. Interferon (IFN)-c release assays (IGRAs) are in vitro tests approved for the detection of LTBI and are based on the measure of IFN-c secreted by T lymphocytes sensitized to Mycobacterium tuberculosis (MTB) antigens. Furthermore, IGRAs provide both a negative and a positive (T-cell mitogen phytohaemagglutinin) control, thus allowing a more comprehensive evaluation of the host immune reactivity. Published evidence suggests that IGRAs maintain their diagnostic sensitivity for LTBI better than the tuberculin skin test (TST) in patients with chronic inflammatory diseases. However, few studies on the performance of IGRAs in patients with psoriasis have been carried out, most of them analysing agreement with TST. The aim of this study was to assess the frequency of LTBI and the host immune reactivity by QuantiFERON-TB Gold InTube (QFT-IT; Qiagen, Hilden, Germany), a commercially available IGRA, in patients with psoriasis and psoriatic arthritis before and during biological treatment. The protocol for this study was approved by the Ethics Committee of the S. OrsolaMalpighi University Hospital. We retrospectively evaluated 188 patients (121 male and 67 female; mean age 50 9 13 0 years, range 18–83; 94 7% Italian born) with mild-to-severe psoriasis (mean Psoriasis Area and Severity Index 13 2 4 9), who were candidates for biological therapy. Fifty-one patients (27 1%) had one or more comorbidities, such as cardiovascular diseases (n = 29), diabetes (n = 17) or obesity (n = 15). Thirty-four (18 1%) were under immunosuppressive therapy at the time of QFT-IT screening, including ciclosporin (n = 20), methotrexate (n = 10) and corticosteroids (n = 4). According to the current recommendations for Italian dermatologists, the screening algorithm included medical history, chest X-ray and QFT-IT, interpreted as positive, negative or indeterminate according to the manufacturer’s instructions. In order to exclude active TB, microbiological standard analysis was performed. A diagnosis of LTBI was based on a positive QFT-IT test in conjunction with no radiological finding suggestive of active TB and negative microbiological assay. For cases of LTBI, a standard prophylactic treatment with isoniazid for 6 months was started 1 month before biological therapy. In case of indeterminate QFT-IT, patients underwent an infectivology consultation and, if necessary, started treatment with isoniazid; these patients were strictly monitored by serial QFT-IT. The study flowchart is depicted in Figure 1. At baseline (t0), 21 patients (11 2%) tested QFT-IT positive and 163 (86 7%) negative; results for four patients (2 1%) were indeterminate, two of whom were under methotrexate treatment. The mean age of patients with positive QFT-IT (59 1 10 4 years) was significantly higher than that of patients with a negative result (48 9 13 5 years) (P = 0 001). After screening, 118 patients (62 8%) started a biological treatment: 106 (89 8%) based on antitumour necrosis factor (anti-TNF)-a blockers (44 etanercept, 44 adalimumab, 18 infliximab), 11 (9 3%) with ustekinumab and one (0 8%) with efalizumab. Concomitantly, most of the patients under immunosuppressive treatment interrupted it. In order to evaluate whether any reversion or conversion could occur during treatment, patients who received biological therapy for longer than 12 months (n = 82) were monitored yearly with QFT-IT for a mean of 25 2 13 2 months. After the first year of biological treatment (t1), 92 7% of patients confirmed the previous QFT-IT result. Out of 10 patients who tested positive for QFT-IT at screening, nine confirmed their positive results during follow-up, maintaining a high level of IFN-c in response to MTB-specific antigens (mean value 6 07 4 93 IU mL 1 at t0, 6 03 5 04 IU mL 1 at t1 and 7 08 5 00 IU mL 1 at t2). In contrast, the only patient with LTBI who showed a reversion at t1 had a baseline QFT-IT value of 0 67 IU mL . Among 68 cases testing QFT-IT negative at screening (t0), two patients experienced a conversion to positive QFT-IT with IFN-c values < 1 IU mL 1 at t1 and t3. Among four patients with an indeterminate result at t0, LTBI was excluded in three of them, who became QFT-IT negative within 3 months, while one patient with risk factors for LTBI started prophylaxis and became QFT-IT positive. A longitudinal analysis of all patients with LTBI (n = 13) treated with isoniazid did not show significant changes in IFN-c response to MTB-specific antigens during biological therapy. To determine whether immune response could be influenced by biological therapy, the trend of IFN-c release in response to QFT-IT mitogen at screening and after 1 year of treatment was analysed. The number of patients with mitogen value