Publications by authors named "Daniela Maria Cirillo"

89 Publications

Performance of the GenoType MTBDRsl V 2.0 for detecting second-line drugs resistance of Mycobacterium tuberculosis isolates in Tunisia.

Res Microbiol 2021 Mar 15:103816. Epub 2021 Mar 15.

National Reference Laboratory for Mycobacteria, A. Mami Pneumology Hospital, Ariana, Tunisia. Electronic address:

Rapid detection of the second-line drug (SLD) resistant tuberculosis (TB) strains is challenging to prescribe an immediate adequate treatment and limit the transmission of SLD resistant strains. The study aimed to evaluate the performance of GenoType MTBDRsl V2.0 compared to phenotypic drug susceptibility testing (pDST:MGIT960) to detect resistance to SLD of Mycobacterium tuberculosis (MTB) isolates in Tunisia, between May 2015 and December 2019. As a matter of fact, 103 rifampicin-resistant and multidrug-resistant MTB strains were included. Discrepancies between pDST and MTBDRsl were solved by whole genome sequencing. Compared to pDST, MTBDRsl V2.0 showed a sensitivity of 92.8% (68.5%-98.7%) in detecting resistance to fluoroquinolones. As for second-line injectable drugs, it presented a sensitivity of 80.0% (49.0%-94.3%). MTBDRsl had sensitivities of 100.0% (67.5%-100.0%), 75.0% (40.9%-92.8%) and 100.0% (60.9%-100.0%) respectively for kanamycin, capreomycin and amikacin. The specificity was 100.0 % for all the drugs evaluated. As for diagnosing XDR-TB, it had a sensitivity of 57.1% (25.0%-84.1%) and a specificity of 100.0% (96.1%-100.0%). MTBDRsl V2.0 showed a high performance in detecting SLD resistance with a short turnaround time compared with pDST, which made it possible to start an early treatment and to maintain a low prevalence of SLD resistance and XDR-TB in Tunisia.
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http://dx.doi.org/10.1016/j.resmic.2021.103816DOI Listing
March 2021

Tackling TB in migrants arriving at Europe's southern border.

Int J Infect Dis 2021 Mar 10. Epub 2021 Mar 10.

Institute for Global Health, University College London, United Kingdom. Electronic address:

Over a quarter of the individuals diagnosed with tuberculosis [TB] in the European Union region are born outside of the area and the proportion has been increasing steadily. Italy is a low TB incidence country with over 50% of TB cases in the foreign-born population primarily due to the high numbers of migrants entering the country via land or sea. As a case study to evaluate the value of screening in newly arrived migrants, the EDETECT-TB project in Italy implemented and evaluated active TB screening in the migrant population at first reception centres to ensure early diagnosis to avoid further spread. Based on a cost-effectiveness analysis from a program provider perspective, a decision tree model allowed the assessment of the value for money of case finding by estimating the cost per case of active TB detected compared with the status quo of no screening. The analysis confirmed that early case detection is a cost-effective intervention in areas with migrants arriving from high TB risk settings. Targeted post-arrival early screening of high TB risk vulnerable new entrants to Italy has a potential role in reducing the spread of TB among migrants.
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http://dx.doi.org/10.1016/j.ijid.2021.02.103DOI Listing
March 2021

Framework for the evaluation of new tests for tuberculosis infection.

Eur Respir J 2021 Jan 21. Epub 2021 Jan 21.

Global TB Programme, World Health Organisation, Geneva, Switzerland.

The scale-up of tuberculosis (TB) preventive treatment (TPT) must be accelerated to achieve the targets set by the United Nations High-level Meeting on TB and the End TB Strategy. The scale-up of effective TPT is hampered by concerns about operational challenges to implement the existing tests for TB infection. New simpler tests could facilitate the scale-up of testing for TB infection. We present a framework for evaluation of new immunodiagnostic tests for the detection of TB infection, with an aim to facilitate their standardised evaluation and accelerate adoption into global and national policies and subsequent scale-up. The framework describes the principles to be considered when evaluating new tests for TB infection and provides guidance to manufacturers, researchers, regulators and other users on study designs, populations, reference standards, sample size calculation and data analysis and it is also aligned with the Global Strategy for TB Research and Innovation adopted by the World Health Assembly in 2020. We also briefly describe technical issues that should be considered when evaluating new tests, including the safety for skin tests, costs incurred by patients and the health system patient, and operational characteristics.
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http://dx.doi.org/10.1183/13993003.04078-2020DOI Listing
January 2021

Repeatability and reproducibility of the wzi high resolution melting-based clustering analysis for Klebsiella pneumoniae typing.

AMB Express 2020 Dec 14;10(1):217. Epub 2020 Dec 14.

Department of Biomedical and Clinical Sciences "L. Sacco", Università di Milano, Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", Milan, Italy.

High resolution melting (HRM) is a fast closed-tube method for nucleotide variant scanning applicable for bacterial species identification or molecular typing. Recently a novel HRM-based method for Klebsiella pneumoniae typing has been proposed: it consists of an HRM protocol designed on the capsular wzi gene and an HRM-based algorithm of strains clustering. In this study, we evaluated the repeatability and reproducibility of this method by performing the HRM typing of a set of K. pneumoniae strains, on three different instruments and by two different operators. The results showed that operators do not affect melting temperatures while different instruments can. Despite this, we found that strain clustering analysis, performed using MeltingPlot separately on the data from the three instruments, remains almost perfectly consistent. The HRM method under study resulted highly repeatable and thus reliable for large studies, even when several operators are involved. Furthermore, the HRM clusters obtained from the three different instruments were highly conserved, suggesting that this method could be applied in multicenter studies, even if different instruments are used.
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http://dx.doi.org/10.1186/s13568-020-01164-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736600PMC
December 2020

Applying a Standardized Approach to Strengthen Performances of GeneXpert Networks programme: lessons learned from Burkina Faso, 2019.

ERJ Open Res 2020 Oct 16;6(4). Epub 2020 Nov 16.

IRCCS San Raffaele Scientific Institute, Milan, Italy.

GeneXpert scale-up is a historic step in the process of tuberculosis (TB) elimination. However, the global roll-out of the test has highlighted gaps that have limited its impact on the TB care cascade. Here we report the description of an innovative GeneXpert network strengthening tool called Applying a Standardized Approach to Strengthen Performances of GeneXpert Networks (ASAP-GxNet) and highlight results and challenges during implementation of the pilot in Burkina Faso. ASAP-GxNet is a 6-month competency-based programme involving an innovative GeneXpert assessment tool as well as a series of short courses and projects designed to qualitatively improve the network while strengthening the capacity of the national GeneXpert focal point to oversee the network. Progress of the GeneXpert network is measured before and at the end of the programme and is rated using a star system (0 to 4 stars). In Burkina Faso, implementation of the ASAP-GxNet programme resulted in improved management of the national GeneXpert network with a 21% increase in points from the start to the end of the programme. To our knowledge, ASAP-GxNet is the first programme to give an overall picture of the quality of GeneXpert networks and to investigate performance in terms of management behaviour. ASAP-GxNet has been developed to help national TB programmes coordinate efforts and needs and highlights the expected achievements of the GeneXpert network.
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http://dx.doi.org/10.1183/23120541.00283-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682669PMC
October 2020

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on Mycobacterium tuberculosis.

Clin Microbiol Infect 2021 Feb 24;27(2):288.e1-288.e4. Epub 2020 Oct 24.

APHP-GHU Nord, Mycobactériologie Spécialisée et de Référence, Laboratoire Associé du Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux (CNR-MyRMA), and Université de Paris, INSERM, IAME UMR1137, F-75006 Paris, France. Electronic address:

Objectives: The first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints.

Methods: During 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD.

Results: Following the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015-0.06) mg/L and 0.12 (0.06-0.25) mg/L for isoniazid, 0.25 mg/L (0.25-0.5) and 0.5 mg/L (0.12-0.5) for levofloxacin, and 0.5 mg/L (0.5-1.0) and 0.5 mg/L (0.5-1.0) for amikacin.

Conclusions: Both SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents.
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http://dx.doi.org/10.1016/j.cmi.2020.10.019DOI Listing
February 2021

Preliminary observations on IGRA testing for TB infection in patients with severe COVID-19 eligible for immunosuppressive therapy.

Respir Med 2020 12 6;175:106204. Epub 2020 Nov 6.

Regional TB Reference Centre and Laboratory, Villa Marelli Institute/ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; Stop TB Italia ONLUS, Milan, Italy.

COVID-19, the novel coronavirus pandemic, has already spread around the globe affecting more than 18 million people. As previously observed with other coronaviruses, SARS-CoV-2 deeply dysregulate the immune system eliciting respiratory failure and a state of systemic hyperinflammation in severely ill individuals. Immunotherapy is often used to downgrade the detrimental effects of the disease sustained by high-level of cytokines. Those treatments, however, are known to undermine patients' ability to contain tuberculosis (TB) infection. This study aims to describe interferon-γ release assay (IGRA) results in severe COVID-19 patients eligible for immunosuppressive treatment. Aggregate data were gathered from five hospitals in Milan, Italy, from March 1 to May 15, 2020 and retrospectively analyses. Results were summarized using absolute frequencies and percentages and compared using a two-sided Chi-squared test. Overall, 462 COVID-19 patients were eligible for immunosuppressive therapy, among which 335 were tested using IGRA testing. More than one-third of them (122/335; 36.4%) had an indeterminate IGRA result because of insufficient immune response to mitogen control, 19 (5.7%) tested positive and 194 (57.9) negative. The majority of patients with lymphocytopenia (i.e., total lymphocyte count [TLC] below 1000 cells/mm) had indeterminate IGRAs (81/155; 52.3%). The proportion becomes even higher in patients with severe lymphocytopenia (i.e., TLC<500 cells/mm) (36/57; 63%). Our results suggest a possible negative impact of COVID-19 related immune dysregulation on TB infection assessment and management. Close monitoring of individuals with or without retesting of individuals with indeterminate IGRAs and further basic science investigations should to be sought to better comprehend their implication on TB epidemiology.
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http://dx.doi.org/10.1016/j.rmed.2020.106204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645275PMC
December 2020

Hepcidin levels predict Covid-19 severity and mortality in a cohort of hospitalized Italian patients.

Am J Hematol 2021 01 3;96(1):E32-E35. Epub 2020 Nov 3.

Regulation of Iron Metabolism Unit-Div, Genetics & Cell Biology-IRCCS San Raffaele Scientific Institute, Milano, Italy.

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http://dx.doi.org/10.1002/ajh.26027DOI Listing
January 2021

Genomic analysis of cardiac surgery-associated Mycobacterium chimaera infections in Italy.

PLoS One 2020 25;15(9):e0239273. Epub 2020 Sep 25.

Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.

One hundred and twenty-two Mycobacterium chimaera strains isolated in Italy from cardiac surgery-related patients, cardiac surgery-unrelated patients and from heater-cooler units, were submitted to whole-genome sequencing and to subsequent SNP analysis. All but one strains isolated from cardiac surgery-related patients belonged to Subgroup 1.1 (19/23) or Subgroup 1.8 (3/23). Only 28 out of 79 strains isolated from heater-cooler units belonged to groupings other than 1.1 and 1.8. The strains isolated from cardiac surgery-unrelated patients were instead distributed across the phylogenetic tree. Our data, the first on isolates from Italy, are in agreement with a recent large genomic study suggesting a common source, represented by strains belonging to Subgroups 1.1 and 1.8, of cardiac surgery-related Mycobacterium chimaera infections. The strains belonging to groupings other than 1.1 and 1.8 isolated from heather-cooler units evidently resulted from contaminations at hospital level and had no share in the Mycobacterium chimaera outbreak. One Mycobacterium chimaera strain investigated in this study proved distant from every previously known Mycobacterium chimaera Groups (1, 2, 3 and 4) and we propose to assign to a novel group, named "Group 5".
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239273PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518601PMC
November 2020

A New Model of Chronic Lung Infection in Immunocompetent Mice.

Int J Mol Sci 2020 Sep 9;21(18). Epub 2020 Sep 9.

Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.

Pulmonary infections caused by (MA) have increased over recent decades, affecting individuals with underlying pathologies such as chronic obstructive pulmonary disease, bronchiectasis and, especially, cystic fibrosis. The lack of a representative and standardized model of chronic infection in mice has limited steps forward in the field of pulmonary infection. To overcome this challenge, we refined the method of agar beads to establish chronic infection in immunocompetent mice. We evaluated bacterial count, lung pathology and markers of inflammation and we performed longitudinal studies with magnetic resonance imaging (MRI) up to three months after infection. In this model, was able to establish a persistent lung infection for up to two months and with minimal systemic spread. Lung histopathological analysis revealed granulomatous inflammation around bronchi characterized by the presence of lymphocytes, aggregates of vacuolated histiocytes and a few neutrophils, mimicking the damage observed in humans. Furthermore, lung lesions were successfully monitored for the first time by MRI. The availability of this murine model and the introduction of the successfully longitudinal monitoring of the murine lung lesions with MRI pave the way for further investigations on the impact of pathogenesis and the efficacy of novel treatments.
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http://dx.doi.org/10.3390/ijms21186590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554715PMC
September 2020

The neglected role of Faith-based Organizations in prevention and control of COVID-19 in Africa.

Trans R Soc Trop Med Hyg 2020 10;114(10):784-786

Centre for Multidisciplinary Research in Health Science, University of Milan, Via Francesco Sforza 35, 20122, Milan, Italy.

The COVID-19 pandemic has exposed health system weaknesses of economically wealthy countries with advanced technologies. COVID-19 is now moving fast across Africa where small outbreaks have been reported so far. There is a concern that with the winter transmission will grow rapidly. Despite efforts of African Governments to promptly establish mitigating measures, rural areas, especially in sub-Saharan Africa, risk being neglected. In those settings, faith-based and other non-governmental organizations, if properly equipped and supported, can play a crucial role in slowing the spread of COVID-19. We describe our experience in two rural health facilities in eSwatini and Ethiopia highlighting the struggle towards preparedness and the urgency of international support to help prevent a major public health disaster.
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http://dx.doi.org/10.1093/trstmh/traa073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499790PMC
October 2020

Advantages and Challenges of Tailored Regimens for Drug-Resistant Tuberculosis: A StopTB Italia Look into the Future.

Infect Drug Resist 2020 11;13:2795-2800. Epub 2020 Aug 11.

StopTB Italia Onlus, Milan 20159, Italy.

The emerge of drug-resistant tuberculosis (TB) strain in recent decades is hampering the efforts of the international community to eliminate the disease worldwide. The World Health Organization (WHO) has drafted many strategies to achieve this ambitious goal. In the very beginning, the aim was to standardize inadequate regimens used in many countries and, thereafter, evolved to tackle the social determinants which hinder TB elimination. However, following the path of narrowing the clinical vision to deal with TB, there is an increased need to personalize the treatment considering both patients and pathogen unique characteristics. In our narrative review, we report the advantages and the backwards in developing a method to implement the concept of precision medicine to the treatment of TB. In this dissertation, we highlight the importance to address different aspects of the diseases encompassing the host and pathogen features, as well as the needs to further implement an adequate follow-up based on the available resources. Nevertheless, many things may hamper the vision of precision medicine in TB, such as the complexity and the costs to develop novel compounds and the costs related to global-scale implementation of patient-centered follow-up. To achieve the ambitious goal of TB elimination, a radical change in TB treatment is needed in order to give a more comprehensive approach based both on patients' peculiarities and driven by drug susceptibility tests and whole-genome sequencing.
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http://dx.doi.org/10.2147/IDR.S257480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429110PMC
August 2020

Emapalumab treatment in an ADA-SCID patient with refractory hemophagocytic lymphohistiocytosis-related graft failure and disseminated bacillus Calmette-Guérin infection.

Haematologica 2021 02 1;106(2):641-646. Epub 2021 Feb 1.

IRCCS San Raffaele Scientific Institute, Milan, Italy.

Emapalumab, a fully human anti-IFNγ monoclonal antibody, has been approved in the US as second-line treatment of primary hemophagocytic lymphohistiocytosis (HLH) patients and has shown promise in patients with graft failure (GF) requiring a second allogeneic hematopoietic stem cell transplantation (HSCT). The blockade of IFNγ activity may increase the risk of severe infections, including fatal mycobacteriosis. We report a case of secondary HLH-related GF in the context of HLA-haploidentical HSCT successfully treated with emapalumab in the presence of concomitant life-threatening infections, including disseminated tuberculosis (TB). A 4 years old girl with Adenosine Deaminase-Severe Combined Immunodeficiency complicated by disseminated TB came to our attention for ex-vivo hematopoietic stem cell-gene therapy. After engraftment failure of gene corrected cells, she received two HLA-haploidentical T-cell depleted HSCT from the father, both failed due to GF related to concomitant multiple infections and secondary HLH. Emapalumab administration allowed to control HLH, as well as to prevent GF after a third haplo-HSCT from the mother. Remarkably, all infections improved with antimicrobial medications and disseminated TB did not show any reactivation. This seminal case supports emapalumab use for treatment of secondary HLH and prevention of GF in patients undergoing haplo-HSCT even in the presence of multiple infections, including TB.
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http://dx.doi.org/10.3324/haematol.2020.255620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849754PMC
February 2021

What is the role of the EUCAST reference method for MIC testing of the Mycobacterium tuberculosis complex?

Clin Microbiol Infect 2020 Nov 6;26(11):1453-1455. Epub 2020 Aug 6.

APHP-GHU Nord site Bichat, Service de Mycobactériologie Spécialisée et de Référence, Laboratoire Associé du Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux (CNR-MyRMA), Paris, France; Université de Paris, INSERM, IAME UMR1137, Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.cmi.2020.07.037DOI Listing
November 2020

Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates - the EUCAST broth microdilution reference method for MIC determination.

Clin Microbiol Infect 2020 Nov 1;26(11):1488-1492. Epub 2020 Aug 1.

APHP-GHU Nord, Mycobactériologie spécialisée et de référence, laboratoire associé du Centre National de référence des mycobactéries et de la résistance des mycobactéries aux antituberculeux (CNR-MyRMA), Paris, France; Université de Paris, INSERM, IAME UMR1137, Paris, France. Electronic address:

Scope: Several methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination for MTBC.

Methods: The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation.

Recommendations: The EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 10 CFU/mL suspension, obtained from a 10 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36° ± 1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 10 CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03-0.12 for isoniazid, 0.12-0.5 for levofloxacin and 0.25-1 mg/L for amikacin.

Conclusions: The EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods will need to be calibrated against this reference method to be used with EUCAST breakpoints.
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http://dx.doi.org/10.1016/j.cmi.2020.07.036DOI Listing
November 2020

Use of a whole genome sequencing-based approach for surveillance in Europe in 2017-2019: an ECDC pilot study.

Eur Respir J 2021 Jan 5;57(1). Epub 2021 Jan 5.

European Centre for Disease Prevention and Control, Stockholm, Sweden.

Whole genome sequencing (WGS) can be used for molecular typing and characterisation of complex (MTBC) strains. We evaluated the systematic use of a WGS-based approach for MTBC surveillance involving all European Union/European Economic Area (EU/EEA) countries and highlight the challenges and lessons learnt to be considered for the future development of a WGS-based surveillance system.WGS and epidemiological data of patients with rifampicin-resistant (RR) and multidrug-resistant (MDR) tuberculosis (TB) were collected from EU/EEA countries between January 2017 and December 2019. WGS-based genetic relatedness analysis was performed using a standardised approach including both core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP)-based calculation of distances on all WGS data that fulfilled minimum quality criteria to ensure data comparability.A total of 2218 RR/MDR-MTBC isolates were collected from 25 countries. Among these, 56 cross-border clusters with increased likelihood of recent transmission (≤5 SNPs distance) comprising 316 RR/MDR-MTBC isolates were identified. The cross-border clusters included between two and 30 resistant isolates from two to six countries, demonstrating different RR/MDR-TB transmission patterns in Western and Eastern EU countries.This pilot study shows that a WGS-based surveillance system is not only feasible but can efficiently elucidate the dynamics of in-country and cross-border RR/MDR-TB transmission across EU/EEA countries. Lessons learnt from this study highlight that the establishment of an EU/EEA centralised WGS-based surveillance system for TB will require strengthening of national integrated systems performing prospective WGS surveillance and the development of clear procedures to facilitate international collaboration for the investigation of cross-border clusters.
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http://dx.doi.org/10.1183/13993003.02272-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784142PMC
January 2021

A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination.

Antimicrob Agents Chemother 2020 08 20;64(9). Epub 2020 Aug 20.

Center for Tuberculosis and WHO Supranational TB Reference Laboratory, National Institute for Communicable Diseases, National Health Laboratory Services, Johannesburg, South Africa.

Criteria defining bedaquiline resistance for tuberculosis have been proposed addressing an emerging concern. We evaluated bedaquiline phenotypic drug susceptibility testing (pDST) criteria using drug-resistant tuberculosis clinical isolates tested at five reference laboratories. Isolates were tested at the proposed bedaquiline MGIT960 and 7H11 agar proportion (AP) critical concentrations and also at higher dilutions. The epidemiological cutoff value for the broth microdilution (BMD) plates (frozen and dry) was investigated. Sanger sequencing was performed ( and genes) for any isolate testing resistant. The composite reference standard (CRS) defined susceptibility or resistance as is if all pDST methods agreed. If the pDST result was discordant, sequencing results were used for final classification. Geographically diverse and bedaquiline-unexposed isolates were tested ( = 495). The epidemiological cutoff value for BMD was confirmed to be 0.12 μg/ml. The majority of isolates were determined to be susceptible by all methods (467/495; 94.3%), and 28 were determined to be resistant by at least one method; 4 of these were determined to be resistant by all methods. Of the 28 resistant isolates, 12 harbored Rv0678 mutations exclusively. Isolates with insertions/deletions were more likely to be determined to be resistant by more than one method (5/7) compared to isolates with a single nucleotide polymorphism (1/5). Applying the CRS to 24 discordant pDST, BMD dry correctly detected most (15/24; 63%), followed by MGIT960 and BMD frozen (13/24; 61%) and lastly AP (12/24; 50%). Applying the CRS, the prevalence of bedaquiline resistance was 2.2% and ranged from 1.4 to 3.4%, depending on the method used. All methods performed well for bedaquiline susceptibility determination; however, resistance detected should be investigated by a second, alternative method.
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http://dx.doi.org/10.1128/AAC.00479-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449194PMC
August 2020

Comparison of core-genome MLST, coreSNP and PFGE methods for cluster analysis.

Microb Genom 2020 04 9;6(4). Epub 2020 Mar 9.

Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.

In this work we compared the most frequently used typing methods: PFGE, cgMLST and coreSNP. We evaluated the discriminatory power of the three methods to confirm or exclude nosocomial transmission on strains isolated from January to December 2017, in the framework of the routine surveillance for multidrug-resistant organisms at the San Raffaele Hospital, in Milan. We compared the results of the different methods to the results of epidemiological investigation. Our results showed that cgMLST and coreSNP are more discriminant than PFGE, and that both approaches are suitable for transmission analyses. cgMLST appeared to be inferior to coreSNP in the CG258 phylogenetic reconstruction. Indeed, we found that the phylogenetic reconstruction based on cgMLST genes wrongly clustered ST258 clade1 and clade2 strains, conversely properly assigned by coreSNP approach. In conclusion, this study provides evidences supporting the reliability of both cgMLST and coreSNP for hospital surveillance programs and highlights the limits of cgMLST scheme genes for phylogenetic reconstructions.
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http://dx.doi.org/10.1099/mgen.0.000347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276701PMC
April 2020

Tools to implement the World Health Organization End TB Strategy: Addressing common challenges in high and low endemic countries.

Int J Infect Dis 2020 Mar 27;92S:S60-S68. Epub 2020 Feb 27.

Directorate General for Disease Surveillance and Control, Ministry of Health, Muscat, Oman; Institute for Clinical Medicine, Faculty of Health Science, University of Aarhus, Denmark; ESCMID Emerging Infections Task Force, Basel, Switzerland.

Aim: The purpose of this viewpoint is to summarize the advantages and constraints of the tools and strategies available for reducing the annual incidence of tuberculosis (TB) by implementing the World Health Organization (WHO) End TB Strategy and the linked WHO TB Elimination Framework, with special reference to Oman.

Methods: The case-study was built based on the presentations and discussions at an international workshop on TB elimination in low incidence countries organized by the Ministry of Health, Oman, which took place from September 5 to September 7, 2019, and supported by the WHO and European Society of Clinical Microbiology and Infectious Diseases (ESCMID).

Results: Existing tools were reviewed, including the screening of migrants for latent TB infection (LTBI) with interferon-gamma release assays, clinical examination for active pulmonary TB (APTB) including chest X-rays, organization of laboratory services, and the existing centres for mandatory health examination of pre-arrival or arriving migrants, including examination for APTB. The need for public-private partnerships to handle the burden of screening arriving migrants for active TB was discussed at length and different models for financing were reviewed.

Conclusions: In a country with a high proportion of migrants from high endemic countries, screening for LTBI is of high priority. Molecular typing and the development of public-private partnerships are needed.
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http://dx.doi.org/10.1016/j.ijid.2020.02.042DOI Listing
March 2020

Whole Genome Sequencing Results Associated with Minimum Inhibitory Concentrations of 14 Anti-Tuberculosis Drugs among Rifampicin-Resistant Isolates of from Iran.

J Clin Med 2020 Feb 7;9(2). Epub 2020 Feb 7.

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran 1417653911, Iran.

Accurate and timely detection of drug resistance can minimize the risk of further resistance development and lead to effective treatment. The aim of this study was to determine the resistance to first/second-line anti-tuberculosis drugs in rifampicin/multidrug-resistant (RR/MDR-MTB) isolates. Molecular epidemiology of strains was determined using whole genome sequencing (WGS)-based genotyping. A total of 35 RR/MDR-MTB isolates were subjected to drug susceptibility testing against first/second-line drugs using 7H9 Middlebrook in broth microdilution method. Illumina technology was used for paired-end WGS applying a Maxwell 16 Cell DNA Purification kit and the NextSeq platform. Data analysis and single nucleotide polymorphism calling were performed using MTBseq pipeline. The genome-based resistance to each drug among the resistant phenotypes was as follows: rifampicin (97.1%), isoniazid (96.6%), ethambutol (100%), levofloxacin (83.3%), moxifloxacin (83.3%), amikacin (100%), kanamycin (100%), capreomycin (100%), prothionamide (100%), D-cycloserine (11.1%), clofazimine (20%), bedaquiline (0.0%), and delamanid (44.4%). There was no linezolid-resistant phenotype, and a bedaquiline-resistant strain was wild type for related genes. The Beijing, Euro-American, and Delhi-CAS were the most populated lineage/sublineages. Drug resistance-associated mutations were mostly linked to minimum inhibitory concentration results. However, the role of well-known drug-resistant genes for D-cycloserine, clofazimine, bedaquiline, and delamanid was found to be more controversial.
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http://dx.doi.org/10.3390/jcm9020465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073636PMC
February 2020

MDR/XDR-TB management of patients and contacts: Challenges facing the new decade. The 2020 clinical update by the Global Tuberculosis Network.

Int J Infect Dis 2020 Mar 4;92S:S15-S25. Epub 2020 Feb 4.

Clinical Epidemiology and Medical Statistics Unit, Department of Medical, Surgical and Experimental Sciences, University of Sassari, Sassari, Italy. Electronic address:

The continuous flow of new research articles on MDR-TB diagnosis, treatment, prevention and rehabilitation requires frequent update of existing guidelines. This review is aimed at providing clinicians and public health staff with an updated and easy-to-consult document arising from consensus of Global Tuberculosis Network (GTN) experts. The core published documents and guidelines have been reviewed, including the recently published MDR-TB WHO rapid advice and ATS/CDC/ERS/IDSA guidelines. After a rapid review of epidemiology and risk factors, the clinical priorities on MDR-TB diagnosis (including whole genome sequencing and drug-susceptibility testing interpretations) and treatment (treatment design and management, TB in children) are discussed. Furthermore, the review comprehensively describes the latest information on contact tracing and LTBI management in MDR-TB contacts, while providing guidance on post-treatment functional evaluation and rehabilitation of TB sequelae, infection control and other public health priorities.
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http://dx.doi.org/10.1016/j.ijid.2020.01.042DOI Listing
March 2020

Accuracy of the QIAxcel Automated System for MIRU-VNTR Genotyping of in Two Limited Resource Settings.

J Clin Med 2020 Feb 1;9(2). Epub 2020 Feb 1.

Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.

Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing of complex (MTBC) isolates, based on 24 loci, is still widely used as the standard for routine molecular surveillance of tuberculosis (TB). QIAxcel system is proposed as an affordable tool that could replace conventional gel electrophoresis and provide high concordance with the reference methods regarding MIRU-VNTR typing of MTBC. We aimed to evaluate the QIAxcel accuracy for allele calling of MIRU-VNTR loci in two regional reference laboratories. A total of 173 DNA were used for the study. Results obtained with QIAxcel were compared to the reference results obtained with an ABI 3730 DNA analyzer. In Albania, the overall agreement with the reference method was 97.92%. A complete agreement result was obtained for 17 loci. In Tunisia, the overall agreement with the reference method was 98.95%. A complete agreement result was obtained for 17 loci. Overall agreement in both centers was 98.43%. In our opinion, use of QIAxcel technology has the potential to be reliable, given an optimized algorithm. Inaccuracies in sizing of long fragments should be solved, especially regarding locus 4052.
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http://dx.doi.org/10.3390/jcm9020389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074178PMC
February 2020

EasyPrimer: user-friendly tool for pan-PCR/HRM primers design. Development of an HRM protocol on wzi gene for fast Klebsiella pneumoniae typing.

Sci Rep 2020 Jan 28;10(1):1307. Epub 2020 Jan 28.

Department of Biomedical and Clinical Sciences "L. Sacco", Università di Milano, Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", Milan, 20157, Italy.

In this work we present EasyPrimer, a user-friendly online tool developed to assist pan-PCR and High Resolution Melting (HRM) primer design. The tool finds the most suitable regions for primer design in a gene alignment and returns a clear graphical representation of their positions on the consensus sequence. EasyPrimer is particularly useful in difficult contexts, e.g. on gene alignments of hundreds of sequences and/or on highly variable genes. HRM analysis is an emerging method for fast and cost saving bacterial typing and an HRM scheme of six primer pairs on five Multi-Locus Sequence Type (MLST) genes is already available for Klebsiella pneumoniae. We validated the tool designing a scheme of two HRM primer pairs on the hypervariable gene wzi of Klebsiella pneumoniae and compared the two schemes. The wzi scheme resulted to have a discriminatory power comparable to the HRM MLST scheme, using only one third of primer pairs. Then we successfully used the wzi HRM primer scheme to reconstruct a Klebsiella pneumoniae nosocomial outbreak in few hours. The use of hypervariable genes reduces the number of HRM primer pairs required for bacterial typing allowing to perform cost saving, large-scale surveillance programs.
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http://dx.doi.org/10.1038/s41598-020-57742-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987216PMC
January 2020

Validation of Bedaquiline Phenotypic Drug Susceptibility Testing Methods and Breakpoints: a Multilaboratory, Multicountry Study.

J Clin Microbiol 2020 03 25;58(4). Epub 2020 Mar 25.

Center for Tuberculosis, National and WHO Supranational TB Reference Laboratory, National Institute for Communicable Diseases, National Health Laboratory Services, Johannesburg, South Africa.

Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious treatments, validated and standardized drug susceptibility testing (DST) is required to improve patient care. This multicountry, multilaboratory external quality assessment (EQA) study aimed to validate the sensitivity, specificity, and reproducibility of provisional bedaquiline MIC breakpoints and World Health Organization interim critical concentrations (CCs) for categorizing clinical isolates as susceptible/resistant to the drug. Three methods were used: Middlebrook 7H11 agar proportion (AP) assay, broth microdilution (BMD) assay, and mycobacterial growth indicator tube (MGIT) assay. Each of the five laboratories tested the 40-isolate (20 unique isolates, duplicated) EQA panel at three time points. The study validated the sensitivity and specificity of a bedaquiline MIC susceptibility breakpoint of 0.12 μg/ml for the BMD method and WHO interim CCs of 1 μg/ml for MGIT and 0.25 μg/ml for the 7H11 AP methods. Categorical agreements between observed and expected results and sensitivities/specificities for correctly identifying an isolate as susceptible/resistant were highest at the 0.25, 0.12, and 1 μg/ml bedaquiline concentrations for the AP method, BMD (frozen or dry plates), and MGIT960, respectively. At these concentrations, the very major error rates for erroneously categorizing an isolate as susceptible when it was resistant were the lowest and within CLSI guidelines. The most highly reproducible bedaquiline DST methods were MGIT960 and BMD using dry plates. These findings validate the use of standardized DST methodologies and interpretative criteria to facilitate routine phenotypic bedaquiline DST and to monitor the emergence of bedaquiline resistance.
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http://dx.doi.org/10.1128/JCM.01677-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098739PMC
March 2020

Prevalence and genetic profiles of isoniazid resistance in tuberculosis patients: A multicountry analysis of cross-sectional data.

PLoS Med 2020 01 21;17(1):e1003008. Epub 2020 Jan 21.

Global TB Programme, World Health Organization, Geneva, Switzerland.

Background: The surveillance of drug resistance among tuberculosis (TB) patients is central to combatting the global TB epidemic and preventing the spread of antimicrobial resistance. Isoniazid and rifampicin are two of the most powerful first-line anti-TB medicines, and resistance to either of them increases the risk of treatment failure, relapse, or acquisition of resistance to other drugs. The global prevalence of rifampicin resistance is well documented, occurring in 3.4% (95% CI 2.5%-4.4%) of new TB patients and 18% (95% CI 7.6%-31%) of previously treated TB patients in 2018, whereas the prevalence of isoniazid resistance at global and regional levels is less understood. In 2018, the World Health Organization (WHO) recommended a modified 6-month treatment regimen for people with isoniazid-resistant, rifampicin-susceptible TB (Hr-TB), which includes rifampicin, pyrazinamide, ethambutol, and levofloxacin. We estimated the global prevalence of Hr-TB among TB patients and investigated associated phenotypic and genotypic drug resistance patterns.

Methods And Findings: Aggregated drug resistance data reported to WHO from either routine continuous surveillance or nationally representative periodic surveys of TB patients for the period 2003-2017 were reviewed. Isoniazid data were available from 156 countries or territories for 211,753 patients. Among these, the global prevalence of Hr-TB was 7.4% (95% CI 6.5%-8.4%) among new TB patients and 11.4% (95% CI 9.4%-13.4%) among previously treated TB patients. Additional data on pyrazinamide and levofloxacin resistance were available from 6 countries (Azerbaijan, Bangladesh, Belarus, Pakistan, the Philippines, and South Africa). There were no cases of resistance to both pyrazinamide and levofloxacin among Hr-TB patients, except for the Philippines (1.8%, 95% CI 0.2-6.4) and Belarus (5.3%, 95% CI 0.1-26.0). Sequencing data for all genomic regions involved in isoniazid resistance were available for 4,563 patients. Among the 1,174 isolates that were resistant by either phenotypic testing or sequencing, 78.6% (95% CI 76.1%-80.9%) had resistance-conferring mutations in the katG gene and 14.6% (95% CI 12.7%-16.8%) in both katG and the inhA promoter region. For 6.8% (95% CI 5.4%-8.4%) of patients, mutations occurred in the inhA promoter alone, for whom an increased dose of isoniazid may be considered. The main limitations of this study are that most analyses were performed at the national rather than individual patient level and that the quality of laboratory testing may vary between countries.

Conclusions: In this study, the prevalence of Hr-TB among TB patients was higher than the prevalence of rifampicin resistance globally. Many patients with Hr-TB would be missed by current diagnostic algorithms driven by rifampicin testing, highlighting the need for new rapid molecular technologies to ensure access to appropriate treatment and care. The low prevalence of resistance to pyrazinamide and fluoroquinolones among patients with Hr-TB provides further justification for the recommended modified treatment regimen.
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http://dx.doi.org/10.1371/journal.pmed.1003008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974034PMC
January 2020

Treating Primary Arthroprosthesis Infection Caused by subsp. a.

Case Rep Infect Dis 2019 14;2019:5892913. Epub 2019 Dec 14.

Infectious Disease Clinic, Department of Medicine, University of Perugia, Perugia, Italy.

Prosthetic joint infections (PJI) caused by nontuberculous mycobacteria are very rare, and results of treatment can be unpredictable. A 72-year-old female underwent hip replacement after an accidental fall in a local hospital in Santo Domingo. The postoperative period was uneventful except for a traumatic wound near the surgical scar. PJI caused by s subsp. was diagnosed 6 months later. A two-stage reimplantation was performed after a 3-month period of aetiology-directed therapy, including amikacin, imipenem, and clarithromycin. . isolate was reported to be resistant to clarithromycin when incubation was protracted for 14 days and to harbour the gene (41). The patient manifested major side effects to tigecycline. At reimplant, microbiologic investigations resulted negative. Overall, medical treatment was continued for a 7-month period. When discontinued and at 6-month follow-up, the patient was clinically well, inflammatory markers were normal, and the radiography showed well-positioned prosthesis. subsp. is a very rare cause of PJI, yet it must be included in the differential diagnosis, especially when routine bacteria cultures are reported being negative. Further investigations are needed to determine any correlations between clinical results and susceptibility tests, as well as the clinical implications of . subsp. harbouring the functional gene (41). Moreover, investigations are needed for determine optimal timings of surgery and lengths of medical therapy to improve patient outcome.
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http://dx.doi.org/10.1155/2019/5892913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942737PMC
December 2019

Towards standardisation: comparison of five whole genome sequencing (WGS) analysis pipelines for detection of epidemiologically linked tuberculosis cases.

Euro Surveill 2019 Dec;24(50)

Tuberculosis Reference Laboratory, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands.

BackgroundWhole genome sequencing (WGS) is a reliable tool for studying tuberculosis (TB) transmission. WGS data are usually processed by custom-built analysis pipelines with little standardisation between them.AimTo compare the impact of variability of several WGS analysis pipelines used internationally to detect epidemiologically linked TB cases.MethodsFrom the Netherlands, 535 complex (MTBC) strains from 2016 were included. Epidemiological information obtained from municipal health services was available for all mycobacterial interspersed repeat unit-variable number of tandem repeat (MIRU-VNTR) clustered cases. WGS data was analysed using five different pipelines: one core genome multilocus sequence typing (cgMLST) approach and four single nucleotide polymorphism (SNP)-based pipelines developed in Oxford, United Kingdom; Borstel, Germany; Bilthoven, the Netherlands and Copenhagen, Denmark. WGS clusters were defined using a maximum pairwise distance of 12 SNPs/alleles.ResultsThe cgMLST approach and Oxford pipeline clustered all epidemiologically linked cases, however, in the other three SNP-based pipelines one epidemiological link was missed due to insufficient coverage. In general, the genetic distances varied between pipelines, reflecting different clustering rates: the cgMLST approach clustered 92 cases, followed by 84, 83, 83 and 82 cases in the SNP-based pipelines from Copenhagen, Oxford, Borstel and Bilthoven respectively.ConclusionConcordance in ruling out epidemiological links was high between pipelines, which is an important step in the international validation of WGS data analysis. To increase accuracy in identifying TB transmission clusters, standardisation of crucial WGS criteria and creation of a reference database of representative MTBC sequences would be advisable.
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http://dx.doi.org/10.2807/1560-7917.ES.2019.24.50.1900130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918587PMC
December 2019