Rapid Detection and Quantification of Mycobacterium tuberculosis DNA in Paraffinized Samples by Droplet Digital PCR: A Preliminary Study.

Background: Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available. Methods: MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert® MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/106 cells. Results: IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values (R 2: 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/106 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550-8,444) copies/106 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples (p = 0.003 and 0.01 for rpoB and p = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease. Conclusion: ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.

Rapid identification of non-tuberculous mycobacteria with MALDI-TOF mass spectrometry.

Rapid and accurate identification of non-tuberculous mycobacteria (NTM) is important for a prompt start to antibiotic therapy. The aim of this study was to obtain accurate identification of NTM quickly by analyzing the performance of the MALDI-TOF mass spectrometry (MS) system VITEK® MS in identifying various NTM species from solid medium and MGIT 960 liquid medium. The study was performed in two phases: preliminary and perspective. Overall, 41/42 species and 33/34 species were correctly identified from the MGIT medium in the preliminary and perspective phases, respectively. The VITEK® MS system includes in its database part of the mycobacteria from the Mycobacterium fortuitum complex but is unable to discriminate among the various species belonging to the complex. Although the VITEK® MS system does not have the protein spectrum of Mycobacterium chimaera, it is not able to distinguish between Mycobacterium chimaera and Mycobacterium intracellulare. Since the VITEK® MS includes the separate protein spectrum of both M. chelonae and M. abscessus, it can discriminate between the two microorganisms. Thanks to these studies we show that the VITEK® MS system is a reliable method for identification of NTMs directly from MGIT liquid medium, instead of the use of solid media.

Prolongation of incubation time improves clinical diagnosis of Mycobacterium xenopi infection and allows susceptibility testing of mycobacterial strains against multiple antibiotics.

OBJECTIVES: Mycobacterium xenopi is a nontuberculous mycobacterium (NTM) whose clinical diagnosis and drug susceptibility studies are frequently hampered by poor in vitro growth. Extending the culture incubation time from 42 days (common-standard) to 56 days could improve the likelihood of diagnosis and provide strains for phenotypic drug susceptibility profiling of this poorly studied but clinically relevant mycobacterium. METHODS: Time-to-positivity of mycobacterial cultures incubated for 56 days were analysed and compared. Clinical mycobacteriosis was defined by ATS/IDSA criteria. In vitro susceptibility of M. xenopi isolates was tested by broth microdilution. RESULTS: Of 3852 mycobacteria-positive cultures (26 different mycobacterial species),M. xenopi required by far the longest growth time in culture, exceeding the 42 days commonly used in routine diagnostics in 41.2% of cases versus 4.7% for other NTM and 2.0% for Mycobacterium tuberculosis complex (P<0.001). Prolonging the incubation time to 56 days had a great impact on M. xenopi diagnosis, as 56.3% (27/48) of patients would have not fulfilled the ATS/IDSA criteria at an incubation limited to 42 days. All 40 M. xenopi isolates from patients with clinical mycobacteriosis were fully susceptibility to macrolides and rifamycins in vitro and to moxifloxacin, amikacin and linezolid. CONCLUSION: These results indicate that a significant percentage (56.3%) of positive culture forM. xenopi would have incorrectly been reported as negative to clinicians without prolonging the incubation time to 56 days. Moreover, 56.3% of patients with M. xenopi disease would have missed the diagnosis along with an appropriate germ-based antimycobacterial treatment, otherwise fully effective.

Large community-acquired Legionnaires' disease outbreak caused by Legionella pneumophila serogroup 1, Italy, July to August 2018.

In July 2018, a large outbreak of Legionnaires' disease (LD) caused by Legionella pneumophila serogroup 1 (Lp1) occurred in Bresso, Italy. Fifty-two cases were diagnosed, including five deaths. We performed an epidemiological investigation and prepared a map of the places cases visited during the incubation period. All sites identified as potential sources were investigated and sampled. Association between heavy rainfall and LD cases was evaluated in a case-crossover study. We also performed a case-control study and an aerosol dispersion investigation model. Lp1 was isolated from 22 of 598 analysed water samples; four clinical isolates were typed using monoclonal antibodies and sequence-based typing. Four Lp1 human strains were ST23, of which two were Philadelphia and two were France-Allentown subgroup. Lp1 ST23 France-Allentown was isolated only from a public fountain. In the case-crossover study, extreme precipitation 5-6 days before symptom onset was associated with increased LD risk. The aerosol dispersion model showed that the fountain matched the case distribution best. The case-control study demonstrated a significant eightfold increase in risk for cases residing near the public fountain. The three studies and the matching of clinical and environmental Lp1 strains identified the fountain as the source responsible for the epidemic.

Real-life evaluation of clinical outcomes in patients undergoing treatment for non-tuberculous mycobacteria lung disease: A ten-year cohort study.

Outcome recognition is a crucial step in the management of non-tuberculous mycobacteria lung disease (NTM-LD). In order to explore NTM-LD outcomes in a real-life setting, an observational, retrospective study enrolling consecutive adults who received treatment for NTM-LD in Milan, Italy, from 2007 to 2017 was conducted. Among 170 patients (68.2% females; median age: 68 years), NTM-LD was mainly due to M. avium complex (MAC) (71.2%), M. kansasii (9.4%) and M. xenopi (7.1%). Along a median follow-up of 31 months, adverse events occurred in 37.6% of the patients. Treatment outcomes of the entire study population included an unsuccessful outcome in 35.3% of the patients, including treatment halted in 13.5%, recurrence in 11.2%, re-infection in 5.3%, treatment failure in 4.1% and relapse in 1.2%. The main reason for treatment halted was drug intolerance. No differences were detected between patients with MAC-LD vs. those with other NTM-LD in terms of unsuccessful outcome in general (35.5% vs. 34.7%). A significantly higher prevalence of patients who underwent treatment halted was found in patients with NTM-LD other than MAC in comparison to patients with MAC-LD (22.4% vs. 9.9%, p = 0.030). One third of adults undergoing treatment for a NTM-LD experiences an unsuccessful outcome with adverse events and treatment discontinuation being major challenges in patients' management.

A rare case of multi-focal human TB after BCG instillation for non-muscle-invasive bladder cancer.

OBJECTIVE: To describe a left epididymitis and para-aortical involvement caused by Mycobacterium tuberculosis hominis reactivation after bacillus Calmette-Guérin instillation for non-muscle-invasive bladder cancer. PATIENT AND METHODS: A Caucasian male, aged 76 years, exposed to bacillus Calmette-Guérin for a high-grade non-muscle-invasive bladder cancer in 2015, reported painful and progressive left scrotal swelling with purulent discharge from a cutaneous fistulous track that yielded, on liquid culture, a pan-susceptible Mycobacterium tuberculosis hominis strain. Moreover, after 6 months of anti-tuberculosis treatment, an abdominal peri-aortic mass, sized 4 cm, was found and a surgical biopsy showed necrotizing granulomas; however, although smear microscopy and Xpert MTB/Rif™ performed on fresh biopsy sample were positive, liquid cultures resulted negative, indicating treatment efficacy. RESULTS: Numerous peculiar and multi-organ involvement due to BCGitis after intravesical immunotherapy have been previously described, including 17 scientific articles about epididymitis, however, no reports so far showed reactivation of Mycobacterium tuberculosis hominis after bacillus Calmette-Guérin treatment. CONCLUSION: Although BCGitis is more prevalent in patients undergoing bacillus Calmette-Guérin instillation for non-muscle-invasive bladder cancer, tuberculosis by other species of Mycobacterium tuberculosis should be always ruled out by molecular and conventional microbiology in patients with a history of Mycobacterium tuberculosis hominis exposure.

Tuberculosis among asylum seekers in Milan, Italy: epidemiological analysis and evaluation of interventions.

In countries of the European Union, tuberculosis (TB) mainly affects marginalised people, including asylum seekers. Migratory flows from high-incidence countries to Italy have increased up to 2017, posing challenges to the national health system. This study sought to assess TB and latent TB infection (LTBI) prevalence among asylum seekers in Milan during the biennium 2016-2017 and to evaluate interventions in place.A two-level active surveillance and screening system was developed for both TB and LTBI. Asylum seekers underwent an initial screening with a tuberculin skin test (TST) and a questionnaire at the receiving sites. At the Regional TB Reference Centre, those with a positive result underwent chest radiography. People aged <35 years with negative chest radiography results underwent further testing by interferon-γ release assay. If results of the assay were positive, LTBI treatment was offered. TB and LTBI prevalence were compared with literature data.A total of 5324 asylum seekers, mostly young (10-39 years; 98%), male (84%) and from sub-Saharan Africa (69%), were enrolled in the study. 69 active TB cases were diagnosed and 863 LTBI-positive individuals were detected. TB prevalence was high (1236 per 100 000 population) and LTBI prevalence was 28%. Despite losses (41%) during the transition from initial screening sites and the diagnostic centre, a good TB cure rate (84%) and optimal LTBI treatment completion (94%) were achieved.Our study shows that TB incidence is high among asylum seekers in Milan and that well-coordinated screening measures are critical for early diagnosis and treatment. It also proves that rolling out successful at-scale interventions for both prophylaxis and disease management is feasible.

Towards tailored regimens in the treatment of drug-resistant tuberculosis: a retrospective study in two Italian reference Centres.

BACKGROUND: The increased incidence of drug-resistant TB is a major challenge for effective TB control. Limited therapeutic options and poor treatment outcomes of DR-TB may increase drug-resistance rates. The objective of the study is to retrospectively compare MDR-TB and pre-XDR-TB treatment regimens and outcomes in two large TB reference centres in Italy from January 2000 to January 2015. METHODS: A retrospective, multicentre study was conducted at the Regional TB Reference Centre Villa Marelli Institute (Milan) and at the Reference Center for MDR-TB and HIV-TB, Eugenio Morelli Hospital (Sondalo). The supra-national Reference Laboratory in Milan performed DST. Inclusion criteria were: age ≥ 18 and culture-confirmed diagnosis of MDR- or pre-XDR TB. Chi-square or Fisher exact test was used to detect differences in the comparison between treatment outcomes, therapeutic regimens, and drug-resistances. Computations were performed with STATA 15. RESULTS: A total of 134 patients were selected. Median (IQR) age at admission was 33 (26-41) years and 90 patients (67.2%) were male. Pulmonary TB was diagnosed in 124 (92.5%) patients. MDR- and pre-XDR-TB cases were 91 (67.9%) and 43 (32.1%), respectively. The WHO shorter MDR-TB regimen could have been prescribed in 16/84 (19.1%) patients. Treatment success was not statistically different between MDR- and pre-XDR-TB (81.3% VS. 81.4%; P = 0.99). Mortality in MDR-TB and pre-XDR-TB groups was 4.4 and 9.3%, respectively (P = 0.2). Median duration of treatment was 18 months and a total of 110 different regimens were administered. Exposure to linezolid, meropenem, and amikacin was associated with a better outcome in both groups (P = 0.001, P < 0.001, and P = 0.004, respectively). CONCLUSIONS: Tailored treatment regimens based on DST results can achieve successful outcomes in patients with pre-XDR-TB.

Performance of real-time PCR Xpert ®MTB/RIF in diagnosing extrapulmonary tuberculosis.

The real time PCR Xpert ® MTB/RIF is fundamental for rapid diagnosis in paucibacillary respiratory samples and for the detection of multidrug-resistant TB cases. This paper aimed to determine its performance on different extrapulmonary samples. We determined sensitivity, specificity, positive and negative predictive value on respiratory and non-respiratory samples collected from January 2010 to June 2014. The protocol for the Xpert ® MTB/RIF PCR suggested by Cepheid was strictly followed for all specimens. In 12257 respiratory samples we observed a sensitivity of 87.1% and a specificity of 99.9%. There were 2818 extrapulmonary specimens, of which 250 were followed by a positive culture for Mycobacterium tuberculosis complex, whereas 72 samples were culture-negative: tuberculosis was clinically confirmed in 71 of them and was excluded for one sample. The sensitivity of the test on urine, pus and CSF samples was 88.2%, 95.6% and 100% respectively. In contrast, the sensitivity of gastric aspirates and biopsies was 81.8% and 83.6% respectively, whereas results of total cavitary fluids were significantly worse than expected (53.7% sensitivity). Our experience shows that Xpert MTB/RIF assay is an accurate, sensitive, and specific test for the rapid detection of pulmonary and extra-pulmonary TB with the only exception of cavitary fluids.

Influence of Hospitalization upon Diagnosis on the Risk of Tuberculosis Clustering.

SETTING: Culture-positive tuberculosis (TB) diagnosed in the metropolitan area of Milan (Italy) over a 5-year period (1995-1999). OBJECTIVE: To assess the impact of short-course hospitalization upon diagnosis on the overall risk of TB clustering. DESIGN: Restriction fragment length polymorphism profiles with a similarity of 100% defined a cluster. Uni- and multivariable logistic regression models were performed to assess factors associated with clustering. RESULTS: Among 1139 patients, 392 (34.4%) were hospitalized before or soon after diagnosis, 405 (35.6%) received domiciliary treatment since the diagnosis and 392 (30%) had no information about initial clinical management. One hundred fifteen molecular clusters involving 363 patients were identified. Using multivariable analysis, hospitalization was not significantly associated with clustering (OR 1.06, 95%CI 0.75-1.50, p=0.575). Subjects aged >65 years old (OR 0.60; 95CI%:0.37-0.95; p=0.016) and non-Italian born patients (OR 0.56; 95%CI:0.41-0.76; p<0.001) were running a lower risk of clustering. Conversely, HIV co-infected patients (OR 1.88, 95%CI:1.20-2.95, p=0.006) and those with MDR TB (OR 2.50, 95%CI:1.46-4.25, p=0.001) were significantly more likely to be involved in clusters. CONCLUSION: In our cohort, domiciliary treatment was not associated with TB clustering. Expanding domiciliary treatment upon diagnosis appears as an advisable measure to reduce unnecessary costs for the health care system.

Tuberculosis outbreak in a primary school, Milan, Italy.

Investigation of an outbreak of tuberculosis (TB) in a primary school in Milan, Italy, found 15 schoolchildren had active TB disease and 173 had latent TB infection. TB was also identified in 2 homeless men near the school. Diagnostic delay, particularly in the index case-patient, contributed to the transmission of infection.

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.