Emanuele Borroni

Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage

Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.

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.

Accuracy of Immunodiagnostic Tests for Active Tuberculosis Using Single and Combined Results: A Multicenter TBNET-Study

Background The clinical application of IFN-γ release assays (IGRAs) has recently improved the diagnosis of latent tuberculosis infection. In a multicenter study of the Tuberculosis Network European Trialsgroup (TBNET) we aimed to ascertain in routine clinical practice the accuracy of a novel assay using selected peptides encoded in the mycobacterial genomic region of difference (RD) 1 for the diagnosis of active tuberculosis in comparison with tuberculin skin test (TST), QuantiFERON-TB GOLD In-Tube (Cellestis Ltd., Carnegie, Australia) and T-SPOT.TB (Oxfordimmunotec, Abingdon, UK). Principal Findings 425 individuals from 6 different European centres were prospectively enrolled. We found that sensitivity of the novel test, TST, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB was respectively 73.1%, 85.3%, 78.1%, and 85.2%; specificity was respectively 70.6%, 48.0%, 61.9% and 44.3%; positive likelihood ratios were respectively 2.48, 1.64, 2.05, and 1.53; negative likelihood ratios were respectively 0.38, 0.31, 0.35, 0.33. Sensitivity of TST combined with the novel test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB increased up to 92.4%, 97.7% and 97.1%, respectively. The likelihood ratios of combined negative results of TST with, respectively, the novel test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB were 0.19, 0.07 and 0.10. Conclusions The assay based on RD1 selected peptides has similar accuracy for active tuberculosis compared with TST and commercial IGRAs. Then, independently of the spectrum of antigens used in the assays to elicit mycobacterial specific immune responses, the novel test, IGRAs, and the TST do not allow an accurate identification of active tuberculosis in clinical practice. However, the combined use of the novel assay or commercial IGRAs with TST may allow exclusion of tuberculosis.

Diagnostic Performance of the New Version (v2.0) of GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study

ABSTRACT Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.

Totally Drug-Resistant and Extremely Drug-Resistant Tuberculosis: The Same Disease?

The emergence of totally drug-resistant (TDR) tuberculosis in India and Iran has been recently discussed in the literature. The 15 TDR tuberculosis cases previously described in Iran were cited as resistant to “all first-line drugs (FLDs) and second-line drugs (SLDs)” [1, 2]. The 4 Mumbai cases were resistant to all FLDs (isoniazid, rifampicin, ethambutol, pyrazinamide, streptomycin) and SLDs tested (ofloxacin, moxifloxacin, kanamycin, amikacin, capreomycin, para-aminosalicylic acid, ethionamide).

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

GenoType MTBDRsl performance on clinical samples with diverse genetic background

We evaluate the performance of the GenoType® MTBDRsl (Hain Lifescience Nehren, Germany) for the detection of second-line resistant tuberculosis and we correlate the frequency of mutations to different Mycobacterium tuberculosis genotypes. We tested 175 strains and 59 clinical specimens interpreting the results according to the Standards for Reporting of Diagnostic Accuracy recommendations. All the strains were also investigated by spoligotyping and Mycobacterial Interspersed Repetitive Units–Variable Number of Tandem Repeats typing. The performances of the MTBDRsl in detecting resistance to fluoroquinolones (FQ), second-line injectable drugs (SLID), and ethambutol (EMB) on clinical isolates were similar (specificity ∼99%, sensitivity ∼70%, and positive predictive value (PPV) ∼99%). Of the 59 respiratory specimens, three samples were classified as “indeterminate”. The specificity in detecting resistances was similar for FQs and EMB 100% (95% CI 92.7–100%) and 100% (95% CI 83.9–100%), respectively with a PPV of 100% (95% CI 64.6–100%) and 100% (95% CI 87.9–100%), respectively. Detection of SLID showed a specificity of 89.1% (95% CI 77.0–95.3%) and a PPV of 58.3% (95% CI 32.0–80.7%). Sensitivity for FQ-resistance detection was 100% (95% CI 64.6–100%), whereas for SLID and EMB it was 89.1% (95% CI 77.0-95.3%) and 86.1% (95% CI 71.3-93.9%), respectively. We detected a significant association between mutations in the rrs gene and Beijing lineage. The MTBDRsl can be used to “rule in” extensively drug-resistant strains of tuberculosis in a high risk group; the low sensitivity and negative predicted value (NPV) make confirmation by conventional drug susceptibility testing mandatory when mutations are not identified. NPV for SLID is higher in Beijing strains, showing that the predictive values of the molecular tests are related to the genetic background.

First evaluation of QuantiFERON-TB Gold Plus performance in contact screening

Identifying latently infected individuals is crucial for the elimination of tuberculosis (TB). We evaluated for the first time the performance of a new type of interferon-γ release assay, QuantiFERON-TB Plus (QFT-Plus), which includes an additional antigen tube (TB2), stimulating both CD4+ and CD8+ T-cells in contacts of TB patients. Contacts were screened for latent TB infection by tuberculin skin test, QFT-Plus and QuantiFERON-TB Gold in Tube (QFT-GIT). In 119 TB contacts, the overall agreement between QFT-Plus and QFT-GIT was high, with a Cohens κ of 0.8. Discordant results were found in 12 subjects with negative QFT-GIT and positive QFT-Plus results. In analyses of markers of TB exposure and test results, the average time spent with the index case was the strongest risk factor for positivity in each of these tests. The difference in interferon-γ production between the two antigen tubes (TB2−TB1) was used as an estimate of CD8+ stimulation provided by the TB2. TB2−TB1 values >0.6 IU·mL−1 were significantly associated with proximity to the index case and European origin. QFT-Plus has a stronger association with surrogate measures of TB exposure than QFT-GIT in adults screened for latent TB infection. Interferon-γ response in the new antigen tube used an indirect estimate of specific CD8+ response correlates with increased Mycobacterium tuberculosis exposure, suggesting a possible role in identifying individuals with recent infection. QuantiFERON-TB Plus improved the diagnostic accuracy for latent TB infection in the setting of contact screening http://ow.ly/2Az0300SDg3

Is multidrug-resistant tuberculosis on the rise in Mozambique? Results of a national drug resistance survey.

To the Editors: Tuberculosis (TB) remains a serious public health problem in many low- and middle-income countries in Africa, Asia and the former Soviet Union [1]. Mozambique is one of them. Despite the fact that in this country, detection of patients with TB has drastically increased during the past decade due to improved notification (from nearly 20,000 new TB cases notified in 1998 to nearly 40,000 in 2008), less than half of the cases of TB estimated to emerge annually are currently being detected. As in other southern African countries, the HIV epidemic is fuelling the TB epidemic in Mozambique, with 60% of patients with TB being co-infected with HIV [1]. Drug-resistant TB is thought to be a major problem, although the latest information available on the magnitude of resistance to anti-TB drugs is from 1998, when the first national drug resistance survey (DRS) was conducted. At that time, multidrug-resistant (MDR)-TB (defined as TB resistant to at least isoniazid and rifampicin, the two main first-line drugs in the treatment of TB) was found among 3.5 and 3.3% of new and previously treated TB cases, respectively [2]. Since then, there has been no further investigation of the magnitude of drug resistance in the country. Based on the latest available data, >3,500 MDR-TB cases are estimated to have emerged in Mozambique in 2008 [3]. Detecting and treating patients with MDR-TB is feasible but substantially more complex and costly than treating patients with fully susceptible Mycobacterium tuberculosis strains. Knowing the …

Drug-resistant tuberculosis among foreign-born persons in Italy

To the Editors: Over the last few years, drug-resistant tuberculosis (TB) has emerged as an important threat to public health in industrialised countries. In Italy, the most recent data on resistance to the first-line drugs (FLDs) streptomycin (S), isoniazid (H), rifampicin (R) and ethambutol (E) were reported for the period 1998–2001 [1]. These studies determined the prevalence of resistance among new cases and previously treated cases, but no information was available on the contribution of immigration, which plays an important role on TB epidemiology in low-incidence countries [2]. In the last decade, while the notified incidence of TB in Italy was stable at approximately seven cases per 100,000 people annually, the proportion of foreign-born persons (FBPs) with TB increased from 22% in 1999 to 46% in 2008 [3]. In the same period, the proportion of African-born persons with TB decreased from 51% to 30%, whereas the proportion of European cases increased from 16% to 33%, most of them being born in Eastern Europe, including Former Soviet Union (FSU) countries. Eastern European countries are among those with the highest TB rates caused by multidrug-resistant (MDR) Mycobacterium tuberculosis strains ( i.e. resistant to at least H and R) and extensively drug-resistant (XDR) strains ( i.e. MDR strains resistant to any fluoroquinolone and to at least one injectable second-line drug (SLD): kanamycin (KM), capreomycin (CM), amikacin (AK)) [4]. Reliable drug susceptibility testing (DST) is essential to diagnose TB caused by drug-resistant strains. In Italy, a network of laboratories coordinated by the World Health Organization (WHO) Supranational Reference Laboratory (SRL) in Rome performs drug susceptibility proficiency testing for S, H, R, E (five rounds from 1997 to 2010) and SLD (KM, AK, CM and ofloxacin (OFL)) (one round in 2010) [5]. In order to understand …