First isolation of Mycobacterium saskatchewanense from medical devices.

Mycobacterium saskatchewanense is a species of pigmented slow-growing Non-Tuberculous Mycobacteria (NTM), positive for Mycobacterium avium complex (MAC) by AccuProbe system. MAC organisms have frequently been isolated from different medical devices. This is the first study reporting isolation of M. saskatchewanense from medical devices and highlights the importance of correctly identifying the NTMs that often colonize sanitary water. GenoType Mycobacterium CM CE-IVD kit (CM) was used as the first step of NTM strain identification, and all positive cultures were found to be components of MAC. Then, GenoType NTM-DR CE-IVD kit (NTM-DR) was used to differentiate the different species. Sub-culture on solid media were used for: (i) phenotypical confirmation by colony morphology and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry; (ii) molecular confirmation by Next Generation Sequencing. All positive cultures were identified as M. intracellulare by CM and NTM-DR assays, whereas colony morphology showed bright yellow scotochromogenic growth. MALDI-TOF analyses identified the strains as M. saskatchewanense with a high score, and identification was confirmed by NGS analysis based on the hsp-65 region. This paper suggests that it is important to actively monitor NTM contamination in medical devices that use sanitary water, to prevent the possibility of patients becoming infected.

Evaluation of trace calls by Xpert MTB/RIF ultra for clinical management in low TB burden settings.

BACKGROUND: Clinical interpretation of trace results by Xpert MTB/RIF Ultra assay (Ultra) used as an initial diagnostic test for tuberculosis (TB) may be challenging. The aim of the study was to evaluate the frequency and epidemiology of trace readouts in routine clinical practice in a low TB prevalence setting and to propose guidance on how to manage patients with trace calls considering the data available (clinical, radiological, bacteriological etc.). MATERIALS AND METHODS: A retrospective, observational, monocentric study was conducted at IRCCS Azienda Ospedaliero-Universitaria of Bologna, Italy between November 2017-December 2020. Presumptive TB patients with at least one Ultra trace result during diagnostic workup before treatment were included in the study. Patients with ongoing anti-TB treatment at the time of the trace call result or with no clinical data available were excluded from the study. RESULTS: Fifty-nine presumptive TB patients with Ultra trace readouts were included in the study (mean age 37.0 years, 61% males). Four patients had a history of TB in the last 2 years. Twenty-five (42.4%) of the 59 samples with trace results were respiratory material. 57/59 (96.6%) patients started anti-TB treatment soon after obtaining trace results, based on clinical, radiological or other information available, while for two patients with a recent history of TB the trace result did not lead to anti-TB treatment. Culture was positive for M. tuberculosis for 31/59 (52.5%) samples with trace calls: 13/25 (52.0%) were respiratory samples and 18/33 (54.5%) non-respiratory samples. The clinical and/or radiological findings of 47/57 (82.4%) patients given anti-TB therapy improved during treatment. CONCLUSION: In low TB incidence settings, Ultra trace calls in presumptive TB patients should be considered as true-positive and treatment should be started promptly, except in cases of recent history of TB, where careful evaluation of other diagnostic criteria is necessary before starting anti-TB treatment. A decisional algorithm for clinical management is proposed.

Use of Fourier-Transform Infrared Spectroscopy With IR Biotyper® System for Legionella pneumophila Serogroups Identification.

Legionella spp. are Gram-negative bacteria that inhabit freshwater environments representing a serious risk for human health. Legionella pneumophila (Lp) is the species most frequently responsible for a severe pneumonia known as Legionnaires' disease. Lp consists of 15 serogroups (Sgs), usually identified by monoclonal or polyclonal antibodies. With regard to Lp serogrouping, it is well known that phenotyping methods do not have a sufficiently high discriminating power, while genotypic methods although very effective, are expensive and laborious. Recently, mass spectrometry and infrared spectroscopy have proved to be rapid and successful approaches for the microbial identification and typing. Different biomolecules (e.g., lipopolysaccharides) adsorb infrared radiation originating from a specific microbial fingerprint. The development of a classification system based on the intra-species identification features allows a rapid and reliable typing of strains for diagnostic and epidemiological purposes. The aim of the study was the evaluation of Fourier Transform Infrared Spectroscopy using the IR Biotyper® system (Bruker Daltonik, Germany) for the identification of Lp at the serogroup (Sg) level for diagnostic purposes as well as in outbreak events. A large dataset of Lp isolates (n = 133) and ATCC reference strains representing the 15 Lp serogroups were included. The discriminatory power of the instrument's classifier, was tested by Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). All isolates were classified as follows: 12/133 (9.0%) as Lp Sg1 and 115/133 (86.5%) as Lp Sg 2-15 (including both ATCC and environmental Lp serogroup). Moreover, a mis-classification for 2/133 (1.5%) isolates of Lp Sg 2-15 that returned as Lp Sg1 was observed, and 4/133 (3.0%) isolates were not classified. An accuracy of 95.49% and an error rate of 4.51% were calculated. IR Biotyper® is able provide a quick and cost-effective reliable Lp classification with advantages compared with agglutination tests that show ambiguous and unspecific results. Further studies including a larger number of isolates could be useful to implement the classifier obtaining a robust and reliable tool for the routine Lp serogrouping. IR Biotyper® could be a powerful and easy-to-use tool to identify Lp Sgs, especially during cluster/outbreak investigations, to trace the source of the infection and promptly adopt preventive and control strategies.

QuantiFERON-TB Gold Plus with Chemiluminescence Immunoassay: Do We Need a Higher Cutoff?

QuantiFERON-TB Gold Plus (QFT-Plus) is the most widely used interferon gamma release assay (IGRA) for the diagnosis of latent tuberculosis infection (LTBI). The aim of this study was to compare QFT-Plus results by enzyme-linked immunosorbent assay (ELISA) on the SkyLab system with those obtained with chemiluminescence immunoassay (CLIA) on the Liaison XL analyzer. Agreement between the two assays was evaluated on 419 QFT-Plus blood samples and was found to be substantial (75.4%); higher agreement was found for positive (95.4%) and negative (80.4%) results, while most discordances were due to ELISA-indeterminate/CLIA-determinate results. According to Italian Clinical Microbiologist Association recommendations, in samples (n = 79) with a borderline result in ELISA (0.20 to 0.70 IU/ml), CLIA median values statistically increased (from 0.29 to 0.59 IU/ml for TB1 and from 0.32 to 0.60 IU/ml for TB2) but remained in the borderline range. Linear regression analysis indicated a substantial correlation between ELISA and CLIA for antigen tubes TB1 (Pearson's r = 0.8666) and TB2 (Pearson's r = 0.8728), but CLIA produced higher values than ELISA. Receiver operating characteristic (ROC) analysis showed that the optimal cutoff value in CLIA was 0.45 IU/ml for TB1 and 0.46 IU/ml for TB2. In conclusion, automated QFT-Plus with CLIA is comparable to QFT-Plus performed by ELISA. Within the linearity range of the test, CLIA detects higher quantitative values than ELISA, resulting in a higher number of determinate results and the conversion of samples that were close to the cutoff into positive borderline results. A higher cutoff for QFT-CLIA needs to be defined based on clinical diagnostic criteria.

Simultaneous detection of Mycobacterium tuberculosis complex and resistance to Rifampicin and Isoniazid by MDR/MTB ELITe MGB® Kit for the diagnosis of tuberculosis.

The MDR/MTB ELITe MGB® Kit on the ELITe InGenius® platform (ELITechGroup SpA, Italy) is the first system for simultaneous detection of the Mycobacterium tuberculosis complex (MTBc) genome and the main mutations responsible for resistance to Isoniazid (inhA, katG) and Rifampicin (rpoB), from decontaminated and heat inactivated samples. In this study we compared the performance of the MDR/MTB ELITe MGB® Kit (ELITe) with culture in 100 pulmonary and 160 extra-pulmonary samples. The sensitivity and specificity of ELITe compared to culture for pulmonary samples were 98.0% and 98.0% respectively; for extra-pulmonary samples the overall sensitivity was 86.3% (80% for urine, 85% for biopsy and gastric aspirate and 95% for cavitary fluid) and specificity was 100%. Genotypic Isoniazid and Rifampicin susceptibility typing was feasible in 96% of sputum MTBc-positive samples and 43% of extra-pulmonary samples; all samples were found to be drug susceptible by phenotypic and ELITe (100% agreement). Detection of mutations in the rpoB, kat G or inhA genes was evaluated on 300 spiked samples (60 per biological matrix) and all resistance profiles were correctly identified by ELITe. Molecular agreement between ELITe and Xpert was 98.0% and 93.3% for pulmonary and extra-pulmonary samples, respectively. In conclusion, our results provide evidence to support the use of MDR/MTB ELITe MGB® Kit in combination with ELITe InGenius® for the diagnosis of MTBc and the detection of Rifampicin and Isoniazid resistance-related mutations in both pulmonary and extra-pulmonary samples. This system simplifies the laboratory workflow, shortens report time and is an aid in choosing appropriate therapeutic treatment and patient management.

Comparison of MycoPrep and the new MYCO-TB kit: rapid and efficient digestion and decontamination of respiratory specimens for the detection of Mycobacteria.

The long incubation time required for Mycobacteria detection may allow cultures to become overgrown by contaminating organisms. Therefore, samples need to be decontaminated before solid and liquid culture. MYCO-TB is a ready-to-use digestion and decontamination kit with single-sample formulation developed by Copan. Sample processing time (3 minutes) is shorter than that of other commercial kits. The aim of this study was to compare the performance of MYCO-TB with MycoPrep, both based on N-acetyl-Lcysteine and sodium hydroxide solution, in terms of culture contamination and Mycobacterial detection by culture. We tested 162 respiratory samples: the overall proportions of contamination of both liquid and solid media were 1.8% for MYCO-TB and 1.8% for MycoPrep. Mycobacterial growth was detected without significant differences in times to positivity (TTP) in liquid culture: 10.5 days for MYCO-TB and 11.1 days for MycoPrep. Samples decontaminated with MYCO-TB were suitable for molecular assays such as Xpert MTB/RIF Ultra and GenoType CMdirect. Extending decontamination times (up to 10 minutes) with MYCO-TB of 20 Mycobacteria-positive specimens did not produce any difference in TTP in liquid culture or in Ultra IS1081/IS6110 probe Ct values. In conclusion, the MYCO-TB kit proved to be effective for the rapid digestion and decontamination of respiratory materials for the detection of Mycobacteria, making it possible to reduce the manual skills required and lower the risk of contamination. Longer decontamination time could be used for samples with a high level of contamination, such as those from cystic fibrosis patients.

Evaluation of MALDI-TOF Mass Spectrometry in Diagnostic and Environmental Surveillance of Legionella Species: A Comparison With Culture and Mip-Gene Sequencing Technique.

Legionella spp. are widespread bacteria in aquatic environments with a growing impact on human health. Between the 61 species, Legionella pneumophila is the most prevalent in human diseases; on the contrary, Legionella non-pneumophila species are less detected in clinical diagnosis or during environmental surveillance due to their slow growth in culture and the absence of specific and rapid diagnostic/analytical tools. Reliable and rapid isolate identification is essential to estimate the source of infection, to undertake containment measures, and to determine clinical treatment. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), since its introduction into the routine diagnostics of laboratories, represents a widely accepted method for the identification of different bacteria species, described in a few studies on the Legionella clinical and environmental surveillance. The focus of this study was the improvement of MALDI-TOF MS on Legionella non-pneumophila species collected during Legionella nosocomial and community surveillance. Comparative analysis with cultural and mip-gene sequencing results was performed. Moreover, a phylogenetic analysis was carried out to estimate the correlations amongst isolates. MALDI-TOF MS achieved correct species-level identification for 45.0% of the isolates belonging to the Legionella anisa, Legionella rubrilucens, Legionella feeleii, and Legionella jordanis species, displaying a high concordance with the mip-gene sequencing results. In contrast, less reliable identification was found for the remaining 55.0% of the isolates, corresponding to the samples belonging to species not yet included in the database. The phylogenetic analysis showed relevant differences inside the species, regruped in three main clades; among the Legionella anisa clade, a subclade with a divergence of 3.3% from the main clade was observed. Moreover, one isolate, identified as Legionella quinlivanii, displayed a divergence of 3.8% from the corresponding reference strain. However, these findings require supplementary investigation. The results encourage the implementation of MALDI-TOF MS in routine diagnostics and environmental Legionella surveillance, as it displays a reliable and faster identification at the species level, as well as the potential to identify species that are not yet included in the database. Moreover, phylogenetic analysis is a relevant approach to correlate the isolates and to track their spread, especially in unconventional reservoirs, where Legionella prevention is still underestimated.

QuantiFERON-TB Performs Better in Children, Including Infants, than in Adults with Active Tuberculosis: a Multicenter Study.

Immunological tests, including the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, represent an important aid for diagnosing active tuberculosis (TB) and latent TB infections in children, but concerns about their use in children <5 years of age persist. This is a multicenter retrospective study comparing a population of 226 children to 521 adults with pulmonary or extrapulmonary TB. The aim was to evaluate the QFT-IT performance, analyzing both qualitative and quantitative results, according to age, birthplace, and disease localization. Compared to culture, QFT-IT sensitivity was 93.9%, 100%, and 94.4% in children ≤2, 2 to 5, and 5 to 16 years of age, respectively, and was significantly higher than that in adults (81.0%) (P < 0.0001). The rate of indeterminate test results for children (2.2%) was significantly lower than that for adults (5.2%) (P < 0.0001). In children, QFT-IT sensitivity was not affected by disease localization or birthplace (Italy born versus foreign born). Interferon gamma (IFN-γ) values in response to TB antigen and mitogen were significantly higher in children than in adults (TB antigen, median of 10 versus 1.66 IU IFN-γ/ml; mitogen, median of 10 versus 6.70 IU IFN-γ/ml; P < 0.0001). In summary, this study supports the use of QFT-IT as a complementary test for the diagnosis of pediatric TB even under 2 years of age. Our observations could be applicable to the new version of the test, QuantiFERON-TB Gold Plus, which has recently been shown to have similar sensitivity in active TB, although data in children are still lacking.

Predictors of time to sputum smear conversion in patients with pulmonary tuberculosis under treatment.

Sputum acid-fast bacilli smear conversion is a fundamental index of treatment response and reduced infectivity in patients with pulmonary tuberculosis (P-TB). To date, there are no models to predict the time to sputum conversion based on patient characteristics. This study aims to ascertain the time to sputum conversion in patients with smear-positive P-TB under treatment, and the variables associated with time to smear conversion. We retrospectively evaluated the time to sputum smear conversion of 89 patients with smear-positive P-TB undergoing treatment at the S. Orsola-Malpighi University Hospital, Bologna (Italy), a referral centre for the diagnosis of TB. Multivariate Cox regression analysis was performed to document variables independently associated with time to conversion. Median time to sputum smear conversion was 24 days (IQR 12-54); the sputum smear converted within the first 2 months of treatment in 78.7% patients. Multivariate Cox regression analysis showed that older age, high baseline mycobacterial load detected by Xpert MTB/RIF, and severity of lung involvement are predictors of persistent smear positivity. The identification of risk factors delaying smear conversion allowed us to develop predictive models that may greatly facilitate the management of smear-positive patients in terms of the duration of respiratory isolation and treatment.

Simkania negevensis in Crohn's Disease.

BACKGROUND: Simkania negevensis is an obligate intracellular Gram-negative bacterium (family Simkaniaceae, order Chlamydiales) that has been isolated from domestic and mains water supplies, is able to infect human macrophages, and can induce an inflammatory response in the host. METHODS: From June to December 2016, in a single-center observational study, colonic Crohn's disease patients and controls (subjects undergoing screening for colorectal cancer) underwent blood tests to identify serum-specific immunoglobulin G (IgG) and immunoglobulin A (IgA) to S. negevensis and a colonoscopy with biopsies for detection of S. negevensis DNA by polymerase chain reaction (PCR). RESULTS: Forty-three Crohn's disease patients and 18 controls were enrolled. Crohn's disease patients had higher prevalence of IgA antibodies to S. negevensis compared with controls (20.9% versus 0%, p = 0.04). Simkaniaceae negevensis DNA was detected in 34.9% and 5.6% of intestinal biopsies in Crohn's disease patients and controls, respectively (p = 0.02). All Crohn's disease patients with PCR-positive biopsies for S. negevensis were IgG seropositive, with specific IgA in 60% of them (p < 0.001). Immunosuppressive therapies, extraintestinal manifestations, or disease activity did not influence the presence of S. negevensis in the Crohn's disease population. CONCLUSIONS: We identified S. negevensis in Crohn's disease patients by demonstrating the presence of S. negevensis mucosal DNA and seropositivity to the bacterium. These results could support the presence of an acute or persistent S. negevensis infection and suggest a possible role in the pathogenesis of Crohn's disease.

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%.

Quantitative Analysis of Gamma Interferon Release Assay Response in Children with Latent and Active Tuberculosis.

The use of interferon gamma (IFN-γ) release assays (IGRAs) for the diagnosis of tuberculosis (TB) infection in children is still under debate because of concerns about the immature immune response in children. The aim of this study was to investigate quantitative values of the QuantiFERON-TB Gold In-Tube (QFT-IT) test, a commercially available IGRA, in a large cohort of children screened for TB infection. A retrospective analysis was conducted on samples from 517 children aged 0 to 14 years old at the Pediatric Unit of S. Orsola-Malpighi University Hospital of Bologna (Italy); quantitative responses to QFT-IT stimuli were analyzed according to diagnosis and age. Elevated IFN-γ values in the QFT-IT nil (background) tube were statistically associated with diagnosis of active TB. Quantitative IFN-γ response to Mycobacterium tuberculosis-specific antigens (TB Ag) was not significantly different in children with active TB compared to those with latent TB infection (LTBI), even though the median values were higher in the first group. When children were grouped by age, those less than 5 years old produced significantly higher levels of IFN-γ in response to TB Ag if they had active TB (median 10 IU/ml) than those with LTBI (median 1.96 IU/ml). IFN-γ response to mitogen increased with age. The overall rate of indeterminate results was low (3.9%), and no indeterminate QFT-IT values were observed in active or latent TB patients. In conclusion, quantitative QFT-IT values could provide further information to clinicians to manage TB in children, and these observations could be transferred to the new version of the test, QuantiFERON-TB Gold Plus, which to date lacks data from the pediatric population.

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.

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.

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.

Performance of interferon-γ release assay for the diagnosis of active or latent tuberculosis in children in the first 2 years of age: a multicenter study of the Italian Society of Pediatric Infectious Diseases.

BACKGROUND: The diagnosis of latent or active tuberculosis in children is often challenging. Recently, interferon-γ release assays have been licensed, but their diagnostic accuracy in young children remains questionable as frequent false-negative or indeterminate results have been reported. METHODS: We performed a multicenter, retrospective study in children 0-24 months of age who were tested at least once with QuantiFERON-TB Gold-in-tube (QTF-IT) ± tuberculin skin test (TST), to analyze its use and performance in clinical practice. RESULTS: Eight-hundred and twenty-three children (449 males, median age 13.5 months) were enrolled. QTF-IT sensitivity and specificity for active tuberculosis were 92.4% and 98.6%, respectively. Indeterminate tests (4.2 %) were not related to age (P = 0.838) or gender (P = 0.223); 32 children (91.4 %) with an indeterminate QTF-IT ultimately resulted uninfected. In the 616 subjects with valid paired results of QTF-IT and TST, sensitivity and specificity were comparable (91.1% vs. 85.1% and 98.1% vs. 97.9%, respectively). Diagnostic concordance between tests was higher in Bacillus Calmétte-Guerin nonvaccinated children (κ = 0.802). A high rate of discordant tests was observed in latent infections. CONCLUSIONS: QTF-IT showed good sensitivity and specificity, and a low rate of indeterminate results in the first 2 years of life, supporting its use at this age. However, considering costs and the similar performance between QTF-IT and TST, it is reasonable to suggest the latter as first-line testing in young children. The complementary use of TST and interferon-γ release assays may be considered in selected cases to improve the accuracy of testing.

Mycobacterium avium subspecies paratuberculosis in the etiology of Crohn's disease, cause or epiphenomenon?

The origin of inflammatory bowel disease is unknown. Attempts have been made to isolate a microorganism that could explain the onset of inflammation, but no pathological agent has ever been identified. Johne's disease is a granulomatous chronic enteritis of cattle and sheep caused by Mycobacterium avium subspecies paratuberculosis (MAP) and shows some analogies with Crohn's disease (CD). Several studies have tried to clarify if MAP has a role in the etiology of CD. The present article provides an overview of the evidence in favor and against the "MAP-hypothesis", analyzing the methods commonly adopted to detect MAP and the role of antimycobacterial therapy in patients with inflammatory bowel disease. Studies were identified through the electronic database, MEDLINE, and were selected based on their relevance to the objective of the review. The presence of MAP was investigated using multiple diagnostic methods for MAP detection and in different tissue samples from patients affected by CD or ulcerative colitis and in healthy controls. On the basis of their studies, several authors support a close relationship between MAP and CD. Although increasing evidence of MAP detection in CD patients is unquestionable, a clear etiological link still needs to be proven.

Trend of microbiologically-confirmed tuberculosis in a low-incidence setting with high immigration rates.

BACKGROUND: The metropolitan area of Bologna, a city in Northern Italy (Emilia Romagna region), is considered a low incidence setting for TB, but has a high rate of foreign immigration (13.5% official resident immigrants relative to the whole population in 2011). The aim of this study was to describe the epidemiological trend of TB, focusing on differences between Italian and foreign-born cases. METHODS: We examined all bacteriologically confirmed TB cases identified in the Microbiology Unit of Bologna University Hospital from January 2008 and December 2011. We compared demographic, clinical and microbiological data for Italian vs. foreign-born TB cases. RESULTS: Out of 255 TB cases identified during the study period, 168 (65.9%) were represented by foreign-born cases. The proportion of immigrants with TB progressively increased over the study period (from 60.8% in 2008 to 67.5% in 2011). Although foreign-born cases were significantly younger than Italian cases (mean age 32.3±14.4 years vs 61.9±21.5 years), the mean age among the latter decreased from 71.2 in 2008 to 54.6 years in 2011 (p=0.036).Concerning TB localization, 65.9% (n=168) had pulmonary TB (P-TB) and 34.1% (n=87) extra-pulmonary TB (EP-TB). In this study, 35.6% of Italian-born P-TB cases were smear positive, versus 51.4% of foreign-born P-TB cases. The highest proportion of high-grade positive microscopy P-TB was among subjects between 25-34 years old (36.9%; p=0.004).Mono-resistance to isoniazid (mono-H) was found among 9.2% and 10.1% of Italian and foreign-born cases, respectively. Among Italian cases, resistance to H and any other first line drug (poly-H) and Multidrug resistant TB (MDR-TB) were 4.6% and 1.2%, respectively. In foreign-born cases poly-H (12.8%) and MDR-TB (6.9%) significantly increased over the time (p=0.003 and p=0.007, respectively). The proportion of MDR-TB was significantly higher among immigrants from Eastern Europe (10.9%) compared to Italian-born patients (p=0.043). All (n=9) MTB strains resistant to four or five first line drugs and Extensively drug resistant (XDR-TB) strains were from foreign-born cases. CONCLUSIONS: TB epidemiology in a low incidence setting is strongly influenced by immigration rates. Ethnicity, mean age, and incidence of MDR-TB among foreign-born cases reflect immigration trends in Northern Italy.

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.