Diagnostic Capacities for Multidrug-Resistant Tuberculosis in the World Health Organization European Region: Action is Needed by all Member States.

The World Health Organization (WHO) recently revised its guidelines for rapid diagnosis of drug-resistant tuberculosis (TB). This study aimed to investigate if TB reference diagnostic services are prepared to support these revisions. An online survey was performed among 44 TB National Reference Laboratories (NRLs) in the WHO European Region. Questions addressed the use of WHO-recommended molecular techniques for the diagnosis of drug-resistant TB, the techniques applied to investigate antimicrobial resistance, and questions on quality assurance. Among 35 of 44 (80%) participating NRLs, 29 of 35 (83%) reported using the GeneXpert platform as the initial test to detect Mycobacterium tuberculosis complex and rifampicin resistance. Five laboratories reported using another WHO-recommended, moderate-complexity, automated nucleic acid amplification test for detection of Mycobacterium tuberculosis complex and resistance to rifampicin and isoniazid. Most (32 of 35; 91%) NRLs reported the capacity to test second-line drugs that have been in clinical use for many years (fluoroquinolones, linezolid, and injectable agents). Only 23 of 35 (66%) and 21 of 35 (60%) NRLs reported the capacity to test bedaquiline and clofazimine. Further efforts will be needed to improve the availability of quality-controlled testing against WHO Group A and Group B drugs. Earlier considerations on the scale-up of diagnostic capacities should be enforced as part of future approval processes for new antimycobacterial agents.

Integrated use of laboratory services for multiple infectious diseases in the WHO European Region during the COVID-19 pandemic and beyond.

Technical advances in diagnostic techniques have permitted the possibility of multi-disease-based approaches for diagnosis and treatment monitoring of several infectious diseases, including tuberculosis (TB), human immunodeficiency virus (HIV), viral hepatitis and sexually transmitted infections (STI). However, in many countries, diagnosis and monitoring, as well as disease response programs, still operate as vertical systems, potentially causing delay in diagnosis and burden to patients and preventing the optimal use of available resources. With countries facing both human and financial resource constraints, during the COVID-19 pandemic even more than before, it is important that available resources are used as efficiently as possible, potential synergies are leveraged to maximise benefit for patients, continued provision of essential health services is ensured. For the infectious diseases, TB, HIV, hepatitis C (HCV) and STI, sharing devices and integrated services starting with rapid, quality-assured, and complete diagnostic services is beneficial for the continued development of adequate, efficient and effective treatment strategies. Here we explore the current and future potential (as well as some concerns), importance, implications and necessary implementation steps for the use of platforms for multi-disease testing for TB, HIV, HCV, STI and potentially other infectious diseases, including emerging pathogens, using the example of the COVID-19 pandemic.

Impact of the COVID-19 pandemic on tuberculosis national reference laboratory services in the WHO European Region, March to November 2020.

We assessed the impact of COVID-19 on diagnostic services for tuberculosis (TB) by national reference laboratories in the WHO European Region. Of 35 laboratories, 30 reported declines in TB sample numbers, amounting up to > 50% of the pre-COVID-19 volumes. Sixteen reported reagent or consumable shortages. Nineteen reallocated ressources to SARS-CoV-2 testing, resulting in an overall increase in workload, largely without a concomitant increase in personnel (n = 14). This poses a risk to meeting the 2025 milestones of the End TB Strategy.

Tuberculosis, HIV, and viral hepatitis diagnostics in eastern Europe and central Asia: high time for integrated and people-centred services.

Globally, high rates (and in the WHO European region an increasing prevalence) of co-infection with tuberculosis and HIV and HIV and hepatitis C virus exist. In eastern European and central Asian countries, the tuberculosis, HIV, and viral hepatitis programmes, including diagnostic services, are separate vertical structures. In this Personal View, we consider underlying reasons for the poor integration for these diseases, particularly in the WHO European region, and how to address this with an initial focus on diagnostic services. In part, this low integration has reflected different diagnostic development histories, global funding sources, and sample types used for diagnosis (eg, typically sputum for tuberculosis and blood for HIV and hepatitis C). Cooperation between services improved as patients with tuberculosis needed routine testing for HIV and vice versa, but financial, infection control, and logistical barriers remain. Multidisease diagnostic platforms exist, but to be used optimally, appropriate staff training and sensible understanding of different laboratory and infection control risks needs rapid implementation. Technically these ideas are all feasible. Poor coordination between these vertical systems remains unhelpful. There is a need to increase political and operational integration of diagnostic and treatment services and bring them closer to patients.

Reducing tuberculosis transmission: a consensus document from the World Health Organization Regional Office for Europe.

Evidence-based guidance is needed on 1) how tuberculosis (TB) infectiousness evolves in response to effective treatment and 2) how the TB infection risk can be minimised to help countries to implement community-based, outpatient-based care.This document aims to 1) review the available evidence on how quickly TB infectiousness responds to effective treatment (and which factors can lower or boost infectiousness), 2) review policy options on the infectiousness of TB patients relevant to the World Health Organization European Region, 3) define limitations of the available evidence and 4) provide recommendations for further research.The consensus document aims to target all professionals dealing with TB (e.g TB specialists, pulmonologists, infectious disease specialists, primary healthcare professionals, and other clinical and public health professionals), as well as health staff working in settings where TB infection is prevalent.

Guiding Principles to Reduce Tuberculosis Transmission in the WHO European Region

Evidence-based guidance is needed on (i) how tuberculosis (TB) infectiousness evolves in response to effective treatment and (ii) how the TB infection risk can be minimized to help countries to implement community-based, outpatient-based care while reducing TB-related suffering and improving TB treatment outcomes. This document aims to 1) review the available evidence on how TB infectiousness evolves in response to effective treatment and which factors can lower or boost infectiousness; 2) present policy options on the infectiousness of TB patients relevant to the WHO European Region; 3) define the limitations in the available evidence and 4) provide recommendations for further research. The document aims to target all professionals dealing with TB (e.g. TB specialists, pulmonologists, infectious disease specialists, primary health care professionals, and other clinical and public health professionals), as well as health staff working insettings where TB transmission may occur.

Hierarchies of host factor dynamics at the entry site of Shigella flexneri during host cell invasion.

Shigella flexneri, the causative agent of bacillary dysentery, induces massive cytoskeletal rearrangement, resulting in its entry into nonphagocytic epithelial cells. The bacterium-engulfing membrane ruffles are formed by polymerizing actin, a process activated through injected bacterial effectors that target host small GTPases and tyrosine kinases. Once inside the host cell, S. flexneri escapes from the endocytic vacuole within minutes to move intra- and intercellularly. We quantified the fluorescence signals from fluorescently tagged host factors that are recruited to the site of pathogen entry and vacuolar escape. Quantitative time lapse fluorescence imaging revealed simultaneous recruitment of polymerizing actin, small GTPases of the Rho family, and tyrosine kinases. In contrast, we found that actin surrounding the vacuole containing bacteria dispersed first from the disassembling membranes, whereas other host factors remained colocalized with the membrane remnants. Furthermore, we found that the disassembly of the membrane remnants took place rapidly, within minutes after bacterial release into the cytoplasm. Superresolution visualization of galectin 3 through photoactivated localization microscopy characterized these remnants as small, specular, patchy structures between 30 and 300 nm in diameter. Using our experimental setup to track the time course of infection, we identified the S. flexneri effector IpgB1 as an accelerator of the infection pace, specifically targeting the entry step, but not vacuolar progression or escape. Together, our studies show that bacterial entry into host cells follows precise kinetics and that this time course can be targeted by the pathogen.

Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time.

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin-3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high-content and high-throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.

Turning on the spotlight--using light to monitor and characterize bacterial effector secretion and translocation.

Secretion and translocation of bacterial pathogen effectors into host cells via dedicated secretion machineries like type III secretion systems (T3SSs) or type IV secretion systems (T4SSs) is a key feature employed by pathogens to attack host cells. Innovative fluorescence and imaging approaches have blossomed during recent years, and became instrumental in revealing the dynamics of effector secretion and function in interfering with host cellular processes, particularly signaling events, gene expression regulation, membrane trafficking, and autophagy. Furthermore, imaging-based screening approaches have demonstrated the mode of action of several bacterial effectors upon host cellular translocation. The rapid technological advancement of imaging technologies indicates that these techniques will continue to be at the center of numerous future breakthroughs delineating the dynamic processes of bacterial effector actions.