Clinical Infectious Diseases

Diagnosing pediatric tuberculosis is highly important due to current challenges posed by nonspecific symptoms. Timely diagnosis enables prompt treatment, reducing morbidity and mortality risks. Prioritizing innovation in pediatric TB diagnostics is essential to address these factors. Tailored diagnostic tools can enhance accuracy, speed, and accessibility of TB diagnosis in children, improving their outcomes. Innovations like collecting bacilli in the breath or using stool and urine to enhance the diagnostic confirmation in children can improve detection and outcomes. Using next generation sequencing for drug susceptibility testing can help in adapting the treatment to the resistance pattern of each child. Finally, following children with TB and analyzing the changes in lung function testing before and after TB treatment can bring much needed knowledge on post TB lung disease in children.

Analysis Borstel TB-Database

Prediction of response to tuberculosis treatment based on prospectively collected data from a clinical reference center in Germany

Until its closure on December 31, 2021, the Borstel Medical Clinic was both an outpatient and inpatient treatment center for people from all over the world suffering from tuberculosis. An extensive database (n = 793) was created from 2011 to 2021 as part of the clinical care of these patients. During their stay in Borstel, patients were regularly examined for the pathogen M. tuberculosis at short intervals using sputum samples, both for diagnostic purposes and during the course of treatment. The amount of microbiological data that could be generated in this way is unique worldwide. Among other things, they make it possible to analyze the time to culture positivity (TTP) during the course of therapy as well as the time to culture conversion (TTC).

The aim of the project is to describe the cohort as a whole using descriptive statistics. This is done using general data (age, gender, origin, ...), comorbidities, risk factors and data on the disease (duration of therapy, therapy outcome, pharmacological resistance, laboratory values, microbiology, ...).

The progression curves of the times to culture positivity up to the final culture conversion are to be presented and analyzed. Correlations and differences between sub-cohorts will be described. These include differences in response to therapy due to gender, pharmacological sensitivity, site of tuberculosis manifestation, time to culture positivity at the time of therapy initiation or CD4+ T-cell count.

A risk score for calculating the mortality associated with tuberculosis will be developed.

In addition, modeling will be used to develop a tool that enables physicians in clinical practice to predict the time of culture conversion of tuberculosis patients.

Detection of Mycobacterium tuberculosis from respiratory masks in adults with pulmonary tuberculosis

Unmask-TB

The aim of the Unmask-TB research project is to develop and test modified FFP2 masks as a diagnostic test for pulmonary tuberculosis in children. Even today, tuberculosis in children can often only be diagnosed clinically, i.e. there is no reliable detection of tuberculous mycobacteria in a laboratory test. In children, failure to recognize tuberculosis is dangerous; up to 40% of children under the age of five die of tuberculosis if undiagnosed and therefore untreated.

Tongue swabs, stool, urine, and the modified masks could be promising new sample types: Obtaining these samples is less burdensome for children compared to conventional methods, such as aspirating gastric liquid. Initial studies also indicate that some of these new methods can better detect tuberculosis in children. At the Borstel Research Centre, we have therefore adapted face masks that have been successfully tested on adults for children. These contain a 3D-printed polyvinyl alcohol strip that is designed to capture bacterial DNA in exhaled breath Our method is also part of the large ‘TB GAPS’ study conducted by Baylor College of Medicine, Houston, USA, in several countries in southern Africa. The German Centre for Infection Research has repeatedly provided us with extensive support in the development of the method.

As part of the EDCTP-funded "CLICK TB" project, new therapy regimes for the treatment of tuberculosis (TB), in particular drug-resistant TB (MDR-TB), are to be investigated. The aim is to clinically test new drug combinations using new active substances. The international consortium includes scientists from Spain and England (GSK), South Africa (TASK, UCT), Norway (University of Tromsø) and Borstel.

• Sanfetrinem cilexetil is a safe oral carbapenem with intrinsic stability against TB β-lactamases. The active substance has already been successfully clinically tested in a phase II study as a broad-spectrum antibiotic. This is the prerequisite for testing sanfetrinem in a phase IIa “Early Bactericidal Activity” study with regard to bactericidal activity in TB.
• GSK3036656 is the first compound in a new class of selective mycobacterial LeuRS inhibitors. The compound has successfully completed the Phase I study

The clinical studies are conducted at an experienced study center in South Africa (TASK) and include extensive biomarker testing (PET-CT and biological markers) to identify new alternatives to traditional culture-based methods for determining the bactericidal activity of a drug or drug combination. The biological biomarkers are measured and analyzed in the Clinical Infectiology laboratory. The tests include, for example, transcriptome analyses and the measurement of transrenal mycobacterial DNA. If the new drugs confirm the hopes, the study consortium aims to quickly transfer the drugs to a phase III study in order to make them available to TB patients and to make a contribution to the fight against TB.

The EDCTP funded project Stool4TB aims to evaluate an innovative stool-based qPCR diagnostic platform, under the hypothesis that it will narrow the extremely large Tuberculosis (TB) case detection gap by improving TB confirmation rates in children and people living with HIV (PLHIV). 

Among children and PLHIV, TB continues to be a leading cause of morbidity and mortality. TB laboratory confirmation is particularly challenging in children and PLHIV given the difficulty in obtaining sputum samples and the paucibacillary nature of the disease.

Another target of Stool4TB is to create a TB diagnostic network among African high TB burden countries (Mozambique, eSwatini, and Uganda) with the capacity to conduct clinical studies of novel diagnostics and new drugs with a focus on children and PLHIV. Furthermore, a biobank of comparative specimens will be created.

As one of the project partners, the Research Center Borstel takes active part in establishing a biobank of samples for future TB-related research. Blood is collected into PAXgene and QuantiFERON-TB Gold (QFT) tubes with the specific goal to validate the TB22 signature and the concept of endotypes in TB in children. RNAseq and QFT data from the studies’ samples will be used to affirm TB22 potential to diagnose TB also in children, to prove its capacity to serve as a marker for outcome prediction in children, to show that it might be capable to serve as a marker for individualized therapy duration in children, to further validate the concept of endotypes in TB, and to perform multi-dimensional analysis in RNAseq and QFT data to further define endotypes in children. PAXgene tubes collected on sites will be shipped to Research Center Borstel where transcriptomic RNA analysis will be performed.

More information: https://www.stool4tb.org/

UNITE4TB (academia and industry united innovation and treatment for tuberculosis) is a public-private partnership consisting of academic institutions, small and medium-sized enterprises (SMEs), public organizations and pharmaceutical companies.

Over a period of seven years (2021-2028), the consortium will be active in around 40 trial sites on four continents (Europe, Asia, Africa and South America) to conduct novel phase 2 clinical trials. These are intended to help accelerate the development of new TB drugs and therapies.

With a financial volume of 180 million euros, UNITE4TB is the largest study in the history of drug development for tuberculosis in history.

The project aims to develop better tolerated drug regimens with shorter treatment duration that can be used to combat tuberculosis with different drug resistance patterns and comorbidities.

Through Professor Lange, the Research Center Borstel is represented in the 4-member steering committee of UNITE4TB. Ms. Ohanna Kirakosyan from the Clinical Infectiology Research Group coordinates the study centers of the consortium in Europe and Mr. Collins Musia is co-leader of the work package on biomarkers. Ms. Nika Zielinski and Dr. Begna Tulu are involved in data analysis and biomarker evaluation.

Further information can be found at: https://www.unite4tb.org/

TBnet e.V. is a German non-governmental organization (NGO) with offices in Riga, Latvia and at the Research Center Borstel. TBnet promotes clinically oriented tuberculosis research in Europe through the exchange and development of ideas and research protocols. It is a European research network that brings together clinicians and researchers in the fight against tuberculosis and aims to improve the quality of care for tuberculosis patients by addressing health inequalities, conducting multicenter clinical trials and training European clinicians and scientists.

In recent years, TBnet has made important contributions to tuberculosis research in the fields of epidemiology, prevention, diagnostics and therapy. A list of TBnet publications can be found here.

TBnet currently has over 640 active members from more than 50 countries (as of May 2024). Ongoing studies focus on improving methods for diagnosis and therapy monitoring, among other things.

Specifically, this involves urinary metabolome analyses, the evaluation of a miniMDR TB test and the improvement of the diagnostic quality of the interferon-gamma release assay (IGRA) in immunosuppressed patients. More general studies are concerned with TB management in organ transplant patients or with the question of the extent to which drug levels in the blood influence specific therapy markers.

Training includes the TBnet Academy, which is aimed at young scientists and doctors to provide them with comprehensive knowledge. In recent years, the TBnet Academy has been held in Athens, Tbilisi and Riga.

TBnet member meetings take place annually on the friday before the ERS conference, where there is an opportunity to exchange ideas, present and discuss the status of projects and initiate new research.

Membership is open to anyone who wishes to make a charitable contribution to tuberculosis research. TBnet does not charge a membership fee

One branch of TBnet is the Nontuberculous Mycobacteria Network (NTMnet). Originally, the Pediatric Tuberculosis Network (pTBnet) was also part of TBnet, but is now independent.

This network emerged in 2011 from the Clinical Infectiology Department of the Research Center Borstel. The current chair is held by Dr. Liga Kuksa from Riga in Latvia. The TBnet secretariat at the Research Center Borstel is headed by Anne Oleischeck (aoleischeck@fz-borstel.de).

Developing a REDCap (Research Electronic Data Capture) database for TBnet research projects is important in order to centralize the data collection and management process and offers customizable data entry forms, facilitating efficient and standardized data entry. Having an operational data collection and management system will provide TBnet with the opportunity of mutualizing research data from different projects and having a more powerful platform for proposals and publications.

Until recently, IGRAs were available in two commercial formats, the ELISPOT-based T-SPOT.TB and the ELISA-based QuantiFERON-TB Gold in-Tube test. Both formats are based on the detection of IFN- g production in T cells or supernatants after stimulation with specific antigens of M. tuberculosis. Among these, the RD-1-encoded proteins ESAT-6 and CFP-10 are most commonly used to identify specific immunity to M. tuberculosis. The peptides derived from these proteins mainly trigger cytokine release from CD4 T cells. Due to the use of M. tuberculosis-specific antigens, in-vitro tests show a higher specificity compared to the skin test. TBnet recently completed a large multicenter study comparing commercial IGRAs and the TST in five different groups of immunocompromised patients. This study showed that IGRAs are superior in detecting LTBI in patient groups with T-cell associated immunodeficiencies, although the positive predictive value (PPV) of IGRAs for the development of tuberculosis was no better than that of the TST. Nevertheless, the percentage of indeterminate results was higher than in non-immunocompromised controls without evidence of TB exposure. In addition, IGRA results were often negative despite the presence of risk factors for exposure to M. tuberculosis and even in patients who developed tuberculosis. However, although IGRAs appear superior to TST for the diagnosis of LTBI, a significant proportion of immunocompromised patients remain undiagnosed, and the tests do not provide a better risk estimate for future development of tuberculosis than TST. Based on the current literature and recent meta-analyses, there is an urgent need for improved in vitro tests and better biomarkers to identify immunocompromised patients with LTBI as a tool to prevent chemotherapy. In response to this need, Qiagen (Hilden, Germany) has developed a next generation ELISA that incorporates the negative and positive control tubes (Nil and Mitogen PHA, respectively) of the current version of the QuantiFERON-TB Gold in tube test (QFT). The next generation QuantiFERON-TB Gold Plus test (QFT-Plus) includes two tubes with M. tuberculosis-specific antigen cocktails (TB1 containing peptides derived from ESAT-6 and CFP-10 (similar to the previously used TB tube). Since this peptide mixture primarily induces the release of cytokines from CD4 T cells, the QFT-Plus assay includes a second M. tuberculosis-specific tube (TB2) containing not only the peptides derived from ESAT-6 and CFP-10, but also additional peptides optimized to stimulate CD8 T cells. Since many immunodeficiencies are associated with either general lymphopenia or specific depletion of CD4 T cells, this additional tube could increase the diagnostic sensitivity for LTBI and active tuberculosis in different groups of immunocompromised patients and reduce the percentage of indeterminacy.

The study will evaluate the performance of this new ELISA-based QuantiFERON-TB plus in-tube test in immunocompromised populations. As in previous TBNET study described (Sester et al. AJRCCM 2014), both qualitative and quantitative test results will be determined with the degree of immunodeficiency and the presence of risk factors for prior exposure to M. tuberculosis. In addition, patients with active tuberculosis (both immunocompetent and immunocompromised individuals) will be included to assess a potential increase in susceptibility in these groups. Immunocompetent individuals at low risk of exposure will be included to assess specificity. Individuals enrolled in this study will be followed for 2 years to assess the predictive value of a positive blood test for progression to active disease longitudinally.

In some countries, patients who receive antituberculosis treatment use adjunctive medicines (“hepatoprotective drugs”) to “protect” the liver from drug injury.

We wish to evaluate and map the prevalence of the use of “hepatoprotective drugs” for patients who receive antituberculosis treatments in different countries of the WHO Europe Region.

The main aim of the study is to investigate common practices in the countries, to find out whether any study has been conducted, costs related to the use of hepatoprotective drugs and their observations on efficacy and safety, and at the end describe and analyse data received.

Global migration has increased in recent decades due to wars, conflicts, persecution, human rights violations and natural disasters, but also due to work or study opportunities. The risk of tuberculosis among migrants depends on the reasons for migration, socio-economic status, the nature of the journey and the risk of tuberculosis in the transit country, as well as the healthcare in the country of origin and the host country.

Despite advancements in TB care for migrants and new treatment strategies, decisions about treatment are often based on expert opinion rather than clinical evidence.

This document summarizes the current state of knowledge on TB diagnosis, TB treatment strategies in migrants, MDR-TB in migrants and HIV/TB co-infection in migrants arriving in Europe. Studies were summarized by meta-analysis when appropriate, otherwise a narrative summary was used.

Based on a systematic review, consensus recommendations were made by experts on all aspects of migrant care.

Once tuberculosis has been diagnosed, anti-tuberculosis therapy is initiated and the course of treatment is monitored regularly over the next six months or more in order to detect treatment failure as early as possible. The most important proof of a treatment response is culture negation, i.e. the absence of growth of mycobacteria in microbiological culture over eight weeks. A molecular biological method for the rapid detection of Mycobacterium tuberculosis in sputum is the amplification of genomic mycobacterial DNA (e.g. GeneXpert®). However, this test system cannot distinguish between dead and viable TB bacteria, meaning that a positive result cannot reliably predict treatment failure. The determination of the expression of replicating genes in the new test system of the molecular tuberculosis bacterial load test (MBLA) has the advantage that only viable bacteria are detected.

In this prospective observational cohort study with Marit Neumann and Prof Dr Christoph Hölscher (Infection Immunology), the accuracy of TB-MBLA will be evaluated for the first time in assessing bacterial load to monitor treatment response in adults with pulmonary MDR/XDR tuberculosis. TB-MBLA results are compared with the GeneXpert method and time to culture positivity (TTP) in Mycobacterial Growth Indicator Tube (MGIT) culture.

In the 10 years from 2012 to 2021, we have treated over 800 tuberculosis patients as inpatients, of which approx. 150 patients with multidrug-resistant tuberculosis (MDR-TB) and approx. 30 patients with extensively drug-resistant tuberculosis (XDR-TB). Data necessary for the correct diagnosis and successful treatment of individual patients was routinely collected in our clinic. All patient data was documented in accordance with the legal requirements of the Data Protection Regulation.

Data from the clinical laboratory, molecular biology or microbiology were recorded at close intervals in order to characterize changes in the quantity and antibiotic sensitivity of the tuberculosis bacteria and to document the course of the tuberculosis disease and the response to therapy. This enabled us to react quickly to any changes. In addition, information about the therapy (medication, dosage, operations if necessary), any side effects and the success of the therapy was recorded. In this way, we try to identify risk factors for delayed treatment response or treatment failure, among other things.

The data collected in everyday clinical practice was compiled in a database, greatly simplifying data collection for clinical studies and enabling additional scientific evaluations. The database was used to produce case studies on individual Borstel patients, e.g. on MDR silicotuberculosis in a quarry worker and on a patient with extremely resistant XDR tuberculosis. An interim evaluation of the database showed that tuberculosis bacteria survived significantly longer in active and former smokers than in kidney smokers.

The data from the database has been used in various completed and ongoing studies, e.g.

• Long-term treatment results for MDR tuberculosis
• MDR tuberculosis treatment based on DNA sequencing
• Port infections during intravenous outpatient therapy
• Prediction of linezolid side effects
• in the DZIF Biomarker Research project
• in the DZIF Therapeutic Drug Monitoring project
• in the Cluster of Excellence Precision Medicine: Individualized therapy of chronic lung infections
• in the TBnet study Management of XDR-TB in Europe
• in a global systematic review on treatment outcomes in children and adolescents with MDR tuberculosis (BENEFIT Kids)

Source: H. Horn und W. Thom "Carl Flügge (1847-1923) Integrator der Hygiene", mhp-Verlag GmbH Wiesbaden 1992, ISBN 3-88681-021-6 Carl Flügge (1847-1923) is best known for his studies on the transmission of tuberculosis and other diseases by droplet infection. Less well known is his work in various areas of applied hygiene. He emphasized the importance of the environment for the development and spread of diseases and investigated various aspects of public health care, from basic microbiological research in the laboratory to housing, nutrition, drinking water supply, sewage disposal and socio-economic and climatic conditions. Early on in his career, he promoted breastfeeding as a remedy for gastrointestinal infections, which were often fatal for infants at the time. He later recommended shielding coughs to protect against tuberculosis and other respiratory infections - advice that became particularly relevant again during and after the coronavirus pandemic.

Carl Flügge was born into a family of doctors in Hanover in 1847 and studied medicine in Göttingen together with Robert Koch. He spent the main years of his academic career at the University of Breslau before accepting the post as Director of the Berlin Institute of Hygiene in 1909. At the time of Carl Flügge's death in 1923, scientific methods and knowledge in the field of hygiene had become so complex that no single person could investigate and survey such a wide range of hygiene-related topics as Carl Flügge did in his work. Carl Flügge was the last great hygienist and founder of environmental medicine as a branch of hygiene.

To mark the 100th anniversary of the death of this great scientist and practitioner, an overview of the life and work of Carl Flügge was written in collaboration with Professor Peter R. Donald from the University of Stellenbosch (South Africa).

The treatment of tuberculosis is becoming increasingly difficult due to increasing antibiotic resistance. The WHO defines MDR (multidrug-resistant) tuberculosis as resistance to rifampicin and isoniazid. XDR (extensively drug-resistant) tuberculosis occurs when there is also resistance to a fluoroquinolone and one of the injectable second-line antibiotics.

As the therapy lasts several months, intravenous therapy must be continued in an outpatient setting. A port is inserted and the patient is trained to administer the medication independently. Prolonged outpatient intravenous therapy by the patient carries risks. The aim of this study is to detect adverse side effects/events, in particular port infection, in this outpatient therapy. Furthermore, the therapy outcome and the therapy costs were included in the evaluation of the therapy success. Patients with MDR, pre-XDR and XDR tuberculosis were included in the study who continued i.v. medication on an outpatient basis and obtained their medication from the Aposan pharmacy. The duration of medication administration and the doses administered until the occurrence of any side effects and port infections as well as the therapy outcome were analyzed.

The data was collected retrospectively. A total of 73 patients whose therapy began between 01/2015 and 12/2020 were included.

Patients whose intravenous therapy ended after discharge were excluded. Furthermore, supply by another pharmacy (e.g. due to moving abroad, etc.) led to exclusion.

Drug-resistant (DR) tuberculosis (TB) cases pose a challenge in the care for TB patients and remain a significant epidemiological issue. Globally, rifampicin- and multidrug-resistant (RR/MDR) cases caused an estimated 160 000 deaths in 2022. In low-incidence countries and regions, foreign-born individuals are often more commonly affected by DR TB. 

Ukraine is one of the countries in the European region with the highest incidence of tuberculosis (TB) and is also globally one of the countries with the highest burden of drug-resistant TB (1 - 3). Further, Ukraine has recently been affected by two unprecedented challenges to the national healthcare system with the COVID-19 pandemic and the outbreak of an armed conflict. The annually reported notification rates for infections caused by Mycobacterium tuberculosis across the European Union and the European Economic Area (EU/EEA) have been gradually declining in the last decades. In 2022, both the total TB notification rates (49.3 versus 8.0/ 100 000) and the proportion of DR TB among bacteriologically confirmed pulmonary cases (4.9% versus 28.3%) in the EU/EEA were substantially lower than the level reported in Ukraine. As of December 2023, about 5 million displaced Ukrainians had received temporary protection status in the countries of the EU/EEA. As a result of this substantial population size increase, the total number of notified TB cases in Ukrainians in the region rose almost four-fold in 2022. The proportion of the DR TB cases of Ukrainian origin amounted to almost 20% of all DR TB cases in the region and over 30% of the laboratory confirmed Ukrainian TB cases were affected by DR TB . In this context we aim to explore the DR TB epidemiology in Ukrainian and other foreign-born TB cases notified in the EU/EEA.

The treatment of patients with drug-resistant tuberculosis is characterized by the number and type of antibiotics and the long duration of therapy, which influence the diversity and composition of the human microbiota. Second-line anti-TB treatment includes several broad-spectrum antibiotics, in particular the injectable aminoglycosides and carbapenems.

In this study, in collaboration with Dr. Mathias Hauptmann from the Cellular Microbiology Research Group, we are investigating how different second-line anti-TB treatment regimens affect the diversity of the human microbiota in the lung and gut in patients with varying degrees of drug-resistant TB.

DRAMATIC is a prospective, Duration-Randomized, Partially Blinded, Phase 2 Study. The objectives are
1) to demonstrate a defined relationship between duration and the proportion of successful outcomes,
2) to determine which durations of the oral experimental regimen can be expected to be as effective as the current validated (but recently de-prioritized) 9-11-month injectable-containing regimen and
3) to define the safety profile of the experimental regimen.

The study randomizes 220 participants, including adults (18 years of age or older) and children (12-17 years of age) with MDR-TB, to 16, 24, 32 or 40 weeks of treatment with a 5-drug oral experimental regimen (bedaquiline, delamanid, linezolid, levofloxacin and clofazimine).

Four experimental treatment durations allow the study to estimate the efficacy of multiple durations of treatment, maximizing the probability to identify the shortest effective experimental regimen. With this design it can be avoided that the duration selected is either too short (thus not effective enough) or too long (thus incurring excess toxicity). By following patients to observe relapse, important information for the optimal treatment duration in a large Phase 3 trial is gathered.

Many biological markers (including metabolic products, proteins, RNA in sputum, blood or urine) will be analyzed to identify new alternatives to traditional culture-based methods for determining the bactericidal activity of a drug or drug combination to predict treatment failure and relapse.

The study is conducted at trial centers in Philippines and Vietnam and is sponsored by the University of California (UCS), and Novartis, Otsuka and Pfizer as industry collaborators. It involves scientists from USA (Boston University, University of Colorado, UCS, Harvard), Philippines, Vietnam and Borstel.

If a new and more effective standard regimen comes into use by the time DRAMATIC has been completed, DRAMATIC will still be able to identify a duration of the experimental regimen that is non-inferior to the new standard of care. This will facilitate moving promptly to a confirmatory Phase 3 trial.

At the Research Center Borstel, a new biomarker (TB22) was identified that enables the individualization of the duration of tuberculosis therapy. For this purpose, a signature was identified from RNA sequences in the peripheral blood of patients. For further validation, access to blood samples from very well characterized tuberculosis patients from Mozambique, Uganda and eSwatini is available through participation in an  EU project.

The TB22 signature will be applied to transcriptome data to determine whether it can accurately diagnose tuberculosis in African children and PLHIV at the start of treatment. In addition, the dynamics of TB22 during treatment will be assessed to determine if the model can accurately predict the time at which the patient has achieved a relapse-free cure.

Transcriptome data may also enable the discovery of new RNA signatures that are not yet known, such as signatures to predict disease progression, detect active tuberculosis, distinguish between tuberculosis and non-tuberculous lung disease, monitor response to treatment, or predict tuberculosis relapse in early stages.

In a longitudinal cohort, samples were taken at various points during treatment to obtain DNA and epigenetic data, and clinical data on disease severity in the form of culture data and imaging procedures, treatment results, medication and any side effects were collected. These data will be used to determine whether and which TB-relevant RNA expressions identified in previous studies can be attributed to DNA mutations or epigenetic disorders in order to draw conclusions about supplementary epigenetic or immunomodulatory therapy. In addition, it will be investigated whether there is a correlation between epigenetic disorders and the clinical manifestations, therapy results and immune cell activity or with existing concepts of personalized TB therapy (e.g. endotypes). Furthermore, it will be investigated whether the epigenetic profile is altered by certain drugs used in TB therapy.

Antibiotic resistance is making the treatment of tuberculosis increasingly difficult. Cultural methods to determine antibiotic resistance are complex and time-consuming. It usually takes several months between the diagnosis of tuberculosis and the receipt of an antibiogram. New methods of gene sequencing allow rapid and comprehensive information on mutations in the genome of tuberculosis bacteria that are associated with antibiotic resistance. These molecular predictions of antibiotic resistance using targeted sequencing technologies (t-NGS) have been officially recommended by the WHO since summer 2023 for the prediction of antibiotic resistance and the composition of treatment regimens. The Research Center Borstel is significantly involved in this process and the preparation of the WHO recommendations.

Whether the treatment results of patients affected by multidrug-resistant tuberculosis (MDR-TB) can actually be improved by using the new technologies is plausible, but has never been proven in a clinical study.

The Research Center Borstel has entered into a strategic partnership with the University of Cape Town, South Africa, to jointly address these and other questions. Our research group is supporting colleagues from the University of Cape Town (Prof. Dr. Keertan Dheda, MD PhD) in the T3 Study 2024, which is investigating the impact of tNGS technology on treatment outcomes of patients with MDR-TB in South Africa. The first patients were enrolled in spring 2024.

The overall endpoint of the study is to determine the impact of t-NGS information-based treatment regimens on treatment outcomes of patients with MDR-TB.

Phase 1 primary endpoint:
proportion of patients initiated on treatment with ≥4 likely effective drugs within 14 days of diagnosis of rifampicin-resistant TB in each group (n=120).

Phase 2 primary endpoint:
rate of unfavorable outcomes 6 months after treatment initiation in each group (n=240 including phase 1 patients)

Phase 3 primary endpoint:
rate of unfavorable outcomes in each group 12 months after treatment initiation (n=300 including phase 2 patients).

The Xpert MTB/RIF Ultra is a rapid molecular assay in cartridge format, first introduced in 2017 as an upgrade of the Xpert MTB/RIF assay for diagnosing tuberculosis. While trace-positive results may represent detection of residual DNA from prior TB, sample cross-contamination, or laboratory error, they may also represent opportunities to detect active TB disease at an early stage. Stool is a clinical sample recommended by the WHO for the diagnosis of TB in children and gaining increasing evidence for adults. We are analyzing trace results from stool in a case-control study of TB patients paired with healthy controls.

A transcriptome model has been developed at the Borstel Research Centre to monitor treatment response in patients with pulmonary tuberculosis. The model uses the response of the human body to an infection with tuberculous mycobacteria to determine whether someone is infected. It could also be used to determine very precisely when someone has been treated with antibiotics long enough to be cured. This means that treatment durations could one day be adapted to each patient and overall shortened.

The model, which is based on 22 genes (TB22 for short), can also be used for diagnostics. This is particularly interesting for children, as many laboratory methods fail to detect tuberculosis in children. There is then no clear evidence of M. tuberculosis, so the diagnosis can only be made clinically. The aim of this research project is to test whether the TB22 model can help in the diagnosis of children or people with HIV infection. In close collaboration with Baylor College of Medicine, Houston, USA, we have been able to obtain RNA samples from children with tuberculosis in Eswatini in southern Africa. The first samples are currently on their way to Borstel and we are starting the measurements for the TB22 transcriptome model.

2023-2024 INSIGHT Scholars with the Baylor Program PI, Dr. Anna Mandalakas (third from right) at orientation in Washington, DC.

The Integrated Network of Scholars in Global Health Research Training (INSIGHT) consortium comprises four U.S. academic institutions (Baylor College of Medicine’s Global TB Program, University of Alabama at Birmingham, University of Maryland Baltimore, University of Pittsburgh) and their affiliated international institutions in Africa, Asia, and the Americas. The program is sponsored by the Fogarty International Center (FIC) and several collaborating Institutes and Centers at the U.S. National Institutes of Health (NIH) and is one of the seven consortia in the NIH Fogarty Launching Future Leaders in Global Health (LAUNCH) Research Training Program. INSIGHT aims to:

1. Create collaborative, multidisciplinary global health research that integrates cross institutional pairing of mentors
2. Provide one year of mentored research training for 105 scholars
3. Promote and support diversity, equity, and inclusion in global health research training
4. Support the transition of scholars into successful and sustainable research careers

This opportunity supports mentored research training in global health for eligible U.S. doctoral candidates and U.S. and LMIC postdoctoral (MD, MD/PhD, ScD or equivalent degree) fellows. Successful applicants spend 12 months abroad at one of 27 affiliated institutions across 20 countries, where they gain experience conducting research in international settings. LMIC postdocs have short-term training at the affiliated U.S. institution. Trainee research projects are supported by experienced and dedicated mentors with expertise in clinical, public health, laboratory, and implementation research.

More information on the INSIGHT training program can be found on the INSIGHT website.

Background: 
In vitro, animal model and clinical evidence suggests that tuberculosis is not a monomorphic disease, and that host response to tuberculosis is protean with multiple distinct molecular pathways and pathologies (endotypes). We applied unbiased clustering to identify separate tuberculosis endotypes with classifiable gene expression patterns and clinical outcomes.

Methods: 
A cohort comprised of microarray gene expression data from microbiologically confirmed tuberculosis patients was used to identify putative endotypes. One microarray cohort with longitudinal clinical outcomes was reserved for validation, as were two RNA-sequencing (seq) cohorts. Finally, a separate cohort of tuberculosis patients with functional immune responses was evaluated to clarify stimulated from unstimulated immune responses.

Results: 
A discovery cohort, including 435 tuberculosis patients and 533 asymptomatic controls, identified two tuberculosis endotypes. Endotype A is characterised by increased expression of genes related to inflammation and immunity and decreased metabolism and proliferation; in contrast, endotype B has increased activity of metabolism and proliferation pathways. An independent RNA-seq validation cohort, including 118 tuberculosis patients and 179 controls, validated the discovery results. Gene expression signatures for treatment failure were elevated in endotype A in the discovery cohort, and a separate validation cohort confirmed that endotype A patients had slower time to culture conversion, and a reduced cure rate. These observations suggest that endotypes reflect functional immunity, supported by the observation that tuberculosis patients with a hyperinflammatory endotype have less responsive cytokine production upon stimulation.

Conclusion: 
These findings provide evidence that metabolic and immune profiling could inform optimisation of endotype-specific host-directed therapies for tuberculosis.

doi: 10.1183/13993003.02263-2021D

Development of a tuberculosis therapeutic drug monitoring platform

The treatment of drug-resistant Mycobacterium tuberculosis relies on complex antibiotic therapy. Inadequate antibiotic exposure can lead to treatment failure, acquired drug resistance, and an increased risk of adverse events. Therapeutic drug monitoring (TDM) can be used to optimize the antibiotic exposure. Therefore, we aimed to develop a single-run multiplex assay using high-performance liquid chromatography-mass spectrometry (HPLC-MS) for TDM of patients with multidrug-resistant, pre-extensively drug-resistant and extensively drug-resistant tuberculosis. A target profile for sufficient performance, based on the intended clinical application, was established and the assay was developed accordingly. Antibiotics were analyzed on a zwitterionic hydrophilic interaction liquid chromatography column and a triple quadrupole mass spectrometer using stable isotope-labeled internal standards. The assay was sufficiently sensitive to monitor drug concentrations over five half-lives for rifampicin, rifabutin, levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, terizidone/cycloserine, ethambutol, delamanid, pyrazinamide, meropenem, prothionamide, and para-amino salicylic acid (PAS). Accuracy and precision were sufficient to support clinical decision making (≤±15% in clinical samples and ±20-25% in spiked samples, with 80% of future measured concentrations predicted to fall within ±40% of nominal concentrations). The method was applied in the TDM of two patients with complex drug-resistant tuberculosis. All relevant antibiotics from their regimens could be quantified and high-dose therapy was initiated, followed by microbiological conversion. In conclusion, we developed a multiplex assay that enables TDM of the relevant first- and second-line anti-tuberculosis medicines in a single run and was able to show its applicability in TDM of two drug-resistant tuberculosis patients.

doi: 10.3390/pharmaceutics15112543

Molecular prediction of treatment regimens in drug -resistant tuberculosis

Background:
Comprehensive and reliable drug susceptibility testing (DST) is urgently needed to provide adequate treatment regimens for patients with multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). We determined whether next-generation sequencing (NGS) analysis of Mycobacterium tuberculosis complex isolates and genes implicated in drug resistance can guide the design of effective MDR/RR-TB treatment regimens.

Methods:
NGS-based genomic DST predictions of M. tuberculosis complex isolates from MDR/RR-TB patients admitted to a TB reference center in Germany between 1 January 2015 and 30 April 2019 were compared with phenotypic DST results of mycobacteria growth indicator tubes (MGIT). Standardized treatment algorithms were applied to design individualized therapies based on either genomic or phenotypic DST results, and discrepancies were further evaluated by determination of minimal inhibitory drug concentrations (MICs) using Sensititre MYCOTBI and UKMYC microtiter plates.

Results:
In 70 patients with MDR/RR-TB, agreement among 1048 pairwise comparisons of genomic and phenotypic DST was 86.3%; 76 (7.2%) results were discordant, and 68 (6.5%) could not be evaluated due to the presence of polymorphisms with yet unknown implications for drug resistance. Importantly, 549 of 561 (97.9%) predictions of drug susceptibility were phenotypically confirmed in MGIT, and 27 of 64 (42.2%) false-positive results were linked to previously described mutations mediating a low or moderate MIC increase. Virtually all drugs (99.0%) used in combination therapies that were inferred from genomic DST were confirmed to be susceptible by phenotypic DST.

Conclusions:
NGS-based genomic DST can reliably guide the design of effective MDR/RR-TB treatment regimens.

doi: 10.1093/cid/ciab359

Long-term treatment outcomes in patients with multidrug-resistant tuberculosis

Objectives:
To describe long-term treatment outcomes in patients with multi-drug-resistant/rifampicin resistant tuberculosis (MDR/RR-TB) and validate established outcome definitions for MDR/RR-TB treatment.

Methods:
Among patients with MDR/RR-TB admitted to a German MDR/RR-TB referral centre from 1 September 2002 to 29 February 2020, we compared long-term treatment outcomes derived from individual patient follow-up with treatment outcomes defined by WHO-2013, WHO-2021 and the Tuberculosis Network European Trials Group-2016.

Results:
In a total of 163 patients (mean age, 35 years; standard deviation, 13 years; 14/163 [8.6%] living with HIV; 109/163 [66.9%] men, 149/163 [91.4%] migrating to Germany within 5 years), the treatment of culture-confirmed MDR/RR-TB was initiated. Additional drug resistance to a fluoroquinolone or a second-line injectable agent was present in 15 of the 163 (9.2%) Mycobacterium tuberculosis strains; resistance against both the drug classes was present in 29 of the 163 (17.8%) strains. The median duration of MDR/RR-TB treatment was 20 months (interquartile range, 19.3-21.6 months), with a medium of five active drugs included. The median follow-up time was 4 years (47.7 months; interquartile range, 21.7 65.8 months). Among the 163 patients, cure was achieved in 25 (15.3%), 82 (50.3%) and 95 (58.3%) patients according to the outcome definitions of WHO-2013, WHO-2021, and the Tuberculosis Network European Trials Group-2016, respectively. The lost to follow-up rate was 17 of 163 (10.4%). Death was more likely in patients living with HIV (hazard ratio, 4.28; 95% confidence interval, 1.26e12.86) and older patients (hazard ratio, 1.08; 95% confidence interval, 1.05e1.12; increment of 1 year). Overall, 101/163 (62.0%) patients experienced long-term, relapse-free cure; of those, 101/122 (82.8%) patients with a known status (not lost to-follow-up or transferred out) at follow-up.

Conclusion:
Under optimal management conditions leveraging individualized treatment regimens, long-term, relapse-free cure from MDR/RR-TB is substantially higher than cure rates defined by current treatment outcome definitions.

doi: 10.1016/j.cmi.2023.02.013

The impact of migration related to the Russia-Ukraine conflict on the epidemiology of tuberculosis infections in the European Union and the European Economic Area

The tuberculosis notification rate in Ukraine is around six times higher than the overall notification rate in the European Union (EU) and the European Economic Area (EEA). Ukraine is also a high priority country for the World Health Organization in terms of antibiotic-resistant tuberculosis. According to Eurostat, around 5 million Ukrainians have been displaced to EU and EEA countries since the beginning of the war between Russia and Ukraine.

In close collaboration with the European Center for Disease Prevention and Control (ECDC), we assessed the epidemiology of tuberculosis in cases of Ukrainian origin reported in the EU/EEA from 2019 to 2022 using routinely collected data.

Our results showed that the number of reported tuberculosis cases among Ukrainians in the region almost quadrupled in 2022 (n = 780, compared to an average of n = 201 in the three previous years). However, cases of Ukrainian origin continued to account for only a small proportion (2.2% in 2022) of all cases reported in the EU/EEA. In addition, notification rates among Ukrainian citizens in the EU/EEA were lower than those reported in Ukraine and remained below 20 per 100,000. The proportion of antibiotic-resistant forms of tuberculosis among cases of Ukrainian origin in the EU/EEA was high, reflecting the high proportion of resistant cases in Ukraine. In 2022, almost one in five antibiotic-resistant TB cases in the EU/EEA was of Ukrainian origin.

These results underline the importance of migrant-sensitive and patient-centered healthcare to ensure early presentation, initiation and continuation of treatment. Due to the high rate of antibiotic-resistant cases, pathogen detection and susceptibility testing in TB patients from Ukraine is crucial.

https://doi.org/10.2807/1560-7917.ES.2024.29.12.2400094

New tuberculosis treatments are needed to address drug resistance, lengthy treatment duration and adverse reactions of available agents. GSK3036656 (ganfeborole) is a first-in-class benzoxaborole inhibiting the Mycobacterium tuberculosis leucyl-tRNA synthetase. Here, in this phase 2a, single-center, open-label, randomized trial, we assessed early bactericidal activity (primary objective) and safety and pharmacokinetics (secondary objectives) of ganfeborole in participants with untreated, rifampicin-susceptible pulmonary tuberculosis. Overall, 75 males were treated with ganfeborole (1/5/15/30 mg) or standard of care (Rifafour e-275 or generic alternative) once daily for 14 days. We observed numerical reductions in daily sputum-derived colony-forming units from baseline in participants receiving 5, 15 and 30 mg once daily but not those receiving 1 mg ganfeborole. Adverse event rates were comparable across groups; all events were grade 1 or 2. In a participant subset, post hoc exploratory computational analysis of 18F-fluorodeoxyglucose positron emission tomography/computed tomography findings showed measurable treatment responses across several lesion types in those receiving ganfeborole 30 mg at day 14. Analysis of whole-blood transcriptional treatment response to ganfeborole 30 mg at day 14 revealed a strong association with neutrophil-dominated transcriptional modules. The demonstrated bactericidal activity and acceptable safety profile suggest that ganfeborole is a potential candidate for combination treatment of pulmonary tuberculosis.

doi: 10.1038/s41591-024-02829-7

Drug-resistant tuberculosis is a substantial health-care concern worldwide. Despite culture-based methods being considered the gold standard for drug susceptibility testing, molecular methods provide rapid information about the Mycobacterium tuberculosis mutations associated with resistance to anti-tuberculosis drugs. This consensus document was developed on the basis of a comprehensive literature search, by the TBnet and RESIST-TB networks, about reporting standards for the clinical use of molecular drug susceptibility testing. Review and the search for evidence included hand-searching journals and searching electronic databases. The panel identified studies that linked mutations in genomic regions of M. tuberculosis with treatment outcome data. Implementation of molecular testing for the prediction of drug resistance in M. tuberculosis is key. Detection of mutations in clinical isolates has implications for the clinical management of patients with multidrug-resistant or rifampicin-resistant tuberculosis, especially in situations when phenotypic drug susceptibility testing is not available. A multidisciplinary team including clinicians, microbiologists, and laboratory scientists reached a consensus on key questions relevant to molecular prediction of drug susceptibility or resistance to M. tuberculosis, and their implications for clinical practice. This consensus document should help clinicians in the management of patients with tuberculosis, providing guidance for the design of treatment regimens and optimising outcomes

doi: 10.1016/S1473-3099(22)00875-1

Background:
European-specific policies for tuberculosis (TB) elimination require identification of key populations that benefit from TB screening.

Aim:
We aimed to identify groups of foreign-born individuals residing in European countries that benefit most from targeted TB prevention screening.

Methods:
The Tuberculosis Network European Trials group (TBnet) collected, by cross-sectional survey, numbers of foreign-born TB patients residing in European Union (EU) countries, Iceland, Norway, Switzerland and the United Kingdom (UK) in 2020 from the 10 highest ranked countries of origin in terms of TB cases in each country of residence. Tuberculosis incidence rates (IRs) in countries of residence were compared with countries of origin.

Results:
Data on 9,116 foreign-born TB patients in 30 countries of residence were collected. Main countries of origin were Eritrea, India, Pakistan, Morocco, Romania and Somalia. Tuberculosis IRs were highest in patients of Eritrean and Somali origin in Greece and Malta (both > 1,000/100,000) and lowest among Ukrainian patients in Poland (3.6/100,000). They were mainly lower in countries of residence than countries of origin. However, IRs among Eritreans and Somalis in Greece and Malta were five times higher than in Eritrea and Somalia. Similarly, IRs among Eritreans in Germany, the Netherlands and the UK were four times higher than in Eritrea.

Conclusions:
Country of origin TB IR is an insufficient indicator when targeting foreign-born populations for active case finding or TB prevention policies in the countries covered here. Elimination strategies should be informed by regularly collected country-specific data to address rapidly changing epidemiology and associated risks.

doi: 10.2807/1560-7917.ES.2023.28.42.2300051

Multidrug-resistant tuberculosis

Tuberculosis (TB) remains the foremost cause of death by an infectious disease globally. Multidrug-resistant or rifampicin-resistant TB (MDR/RR-TB; resistance to rifampicin and isoniazid, or rifampicin alone) is a burgeoning public health challenge in several parts of the world, and especially Eastern Europe, Russia, Asia and sub-Saharan Africa. Pre-extensively drug-resistant TB (pre-XDR-TB) refers to MDR/RR-TB that is also resistant to a fluoroquinolone, and extensively drug-resistant TB (XDR-TB) isolates are additionally resistant to other key drugs such as bedaquiline and/or linezolid. Collectively, these subgroups are referred to as drug-resistant TB (DR-TB). All forms of DR-TB can be as transmissible as rifampicin-susceptible TB; however, it is more difficult to diagnose, is associated with higher mortality and morbidity, and higher rates of post-TB lung damage. The various forms of DR-TB often consume >50% of national TB budgets despite comprising <5-10% of the total TB case-load. The past decade has seen a dramatic change in the DR-TB treatment landscape with the introduction of new diagnostics and therapeutic agents. However, there is limited guidance on understanding and managing various aspects of this complex entity, including the pathogenesis, transmission, diagnosis, management and prevention of MDR-TB and XDR-TB, especially at the primary care physician level.

DOI: 10.1038/s41572-024-00504-2

Impact of bedaquiline on treatment outcome in drug-resistant tuberculosis

Background:
Evaluation of novel anti-tuberculosis (TB) drugs for the treatment of multidrug-resistant (MDR)-TB continues to be of high interest on the TB research agenda. We assessed treatment outcomes in patients with pulmonary MDR-TB who received bedaquiline-containing treatment regimens in the Republic of Moldova, a high-burden MDR-TB country.

Methods:
We systematically analysed the SIMETB national electronic TB database and performed a retrospective propensity score-matched comparison of treatment outcomes in a cohort of patients with MDR-TB who started treatment during 2016-2018 with a bedaquiline-containing regimen (bedaquiline cohort) and a cohort of patients treated without bedaquiline (non-bedaquiline cohort).

Results:
Following propensity score matching, 114 patients were assigned to each cohort of MDR-TB patients. Patients in the bedaquiline cohort had higher 6-month sputum culture conversion rate than those in the non-bedaquiline cohort (66.7% versus 40.3%; p<0.001). Patients under bedaquiline-containing regimens had a higher cure rate assessed by both World Health Organization (WHO) and TBnet definitions (55.3% versus 24.6%; p=0.001 and 43.5% versus 19.6%; p=0.004, respectively), as well as a lower mortality rate (8.8% versus 20.2%; p<0.001 and 10.9% versus 25.2%; p=0.01, respectively). In patients who previously failed on MDR-TB treatment, >40% of patients achieved a cure with a bedaquiline-containing regimen.

Conclusions:
Bedaquiline-based MDR-TB treatment regimens result in better disease resolution when compared with bedaquiline-sparing MDR-TB treatment regimens under programmatic conditions in a country with a high burden of MDR-TB.

doi: 10.1183/13993003.02544-2020

Use of TNF antagonists and JAK inhibitors in patients with previous tuberculosis

Tumor necrosis factor (TNF) antagonists and Janus kinase (JAK) inhibitors are important treatment options for chronic inflammatory diseases such as rheumatoid arthritis. There is currently uncertainty about how to manage patients with prior or current tuberculosis who require treatment with TNF antagonists or JAK inhibitors for underlying inflammatory conditions. As part of a systematic review, we searched the medical literature for corresponding reports and identified 368 patients who were treated with TNF antagonists during or after tuberculosis. Only 14 of these patients (3.8 %) had a recurrence of tuberculosis. Of the eleven patients who received a JAK inhibitor during or after tuberculosis, only one patient (9.1 %) had a recurrence of tuberculosis. These low numbers suggest that the use of TNF antagonists and JAK inhibitors may be relatively safe in patients with current or previous TB and the need for further treatment of underlying diseases

doi: 10.1016/j.cmi.2024.04.011

Consensus management recommendations for less common non-tuberculous mycobacterial pulmonary diseases

The 2020 clinical practice guideline for the treatment of non-tuberculous mycobacterial pulmonary disease (NTM-PD) by the American Thoracic Society, European Respiratory Society, European Society of Clinical Microbiology and Infectious Diseases, and Infectious Diseases Society of America; and the 2017 management guideline by the British Thoracic Society covered pulmonary diseases in adults caused by Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium xenopi, and Mycobacterium abscessus. In order to provide evidence-based recommendations for the treatment of less common non-tuberculous mycobacterial (NTM) species in adult patients without cystic fibrosis or HIV infection, our expert panel group performed systematic literature searches to provide management guidance for pulmonary diseases caused by seven additional organisms: Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium malmoense, Mycobacterium simiae, and Mycobacterium szulgai. Treatment recommendations were developed by a structured consensus process. The evidence from the scientific literature published in English for treatment recommendations for pulmonary diseases caused by other NTM species was of very low quality, with the exception of M malmoense, and based on the evaluation of case reports and case series. For M malmoense, results from two randomised controlled trials and three retrospective cohort studies provided a better evidence base for treatment recommendations, although the evidence was still of low quality.

doi: 10.1016/S1473-3099(21)00586-7

Prediction of adverse events in tuberculosis treatment

Adverse events can make the treatment of tuberculosis more difficult and can be associated with a reduction in the patient's quality of life. The underlying mechanisms are not yet known for all drugs, meaning that adverse events cannot be completely prevented to date.

The aim of this project is therefore to identify and validate gene signatures using machine-learning algorithms to predict severe adverse events before the start of tuberculosis treatment. Transcriptome-based signatures are particularly suitable here. In addition, possible explanatory approaches for the development of severe adverse events are to be generated by identification and biological interpretation of genes and pathways. This would be a possible foundation for further research aimed at gaining a better understanding of the mechanismsand, if necessary, taking preventive countermeasures.

With the help of this work, it will hopefully be possible in the near future to identify an increased risk of serious adverse events before exposure to medication and thus make a contribution to personalised medicine in tuberculosis treatment.

https://doi.org/10.20411/pai.v9i2.705