[Treatment of MDR, pre-XDR, XDR and rifampicin resistant tuberculosis or in case of intolerance to at least rifampicin in Austria, Germany and Switzerland - Amendment dated 19.09.2023 to the Sk2-Guideline: Tuberculosis in adulthood of the German Central Committee against Tuberculosis (DZK) on behalf of the German Respiratory Society (DGP)]. / Therapie bei MDR-, prä-XDR-, XDR-Tuberkulose und Rifampicin-Resistenz oder bei Medikamentenunverträglichkeit gegenüber mindestens Rifampicin.

In December 2022, based on the assessment of new evidence, the World Health Organization (WHO) updated its guidelines for the treatment of drug-resistant tuberculosis (TB). The evaluation of both, these recommendations, and the latest study data, makes it necessary to update the existing guidelines on the treatment of at least rifampicin-resistant tuberculosis for the German-speaking region, hereby replacing the respective chapters. A shortened MDR-TB treatment of at least 6 month using the fixed and non-modifiable drug combination of bedaquiline, pretomanid, linezolid, and moxifloxacin (BPaLM) is now also recommended for Germany, Austria, and Switzerland under certain conditions. This recommendation applies to TB cases with proven rifampicin resistance, including rifampicin monoresistance. For treatment of pre-extensively drug resistant TB (pre-XDR-TB), an individualized treatment for 18 months adjusted to resistance data continues to be the primary recommendation. The non-modifiable drug combination of bedaquiline, pretomanid, and linezolid (BPaL) may be used alternatively in pre-XDR TB if all prerequisites are met. The necessary prerequisites for the use of BPaLM and BPaL are presented in this amendment to the S2k guideline for 'Tuberculosis in adulthood'.

[Tuberculosis Infection Control & Hygiene - Recommendations of the DZK]. / Infektionsprävention & Hygiene bei Tuberkulose ­ Empfehlungen des DZK.

Preventing the spread of the disease is an essential goal in the care and treatment of tuberculosis. In addition to early diagnosis and effective therapies, isolation of infectious patients and adequate hygiene measures are of particular importance for infection prevention. The present recommendations replace the previous recommendations "tuberculosis infection control" from 2012 and take into account the current national and international recommendations and as well as new scientific findings. After a description of the infection and the transmission pathways, the necessary prevention and hygiene measures in health care facilities are comprehensively presented. Since the last revision of the recommendations on infection prevention, international recommendations and the KRINKO recommendation on ending isolation have been changed. In accordance with this, under certain conditions in the case of sensitive tuberculosis, de-isolation in health care facilities can take place after 14 days without taking the sputum findings into account. The second part of the recommendations explains in detail the measures to be taken in special situations and areas, such as general practitioners, ambulance services and care facilities. Here, the recommendations on respiratory protection have been simplified; for staff, an FFP2 mask is now generally considered sufficient.

[Tuberculosis in adulthood - The Sk2-Guideline of the German Central Committee against Tuberculosis (DZK) and the German Respiratory Society (DGP) for the diagnosis and treatment of adult tuberculosis patients]. / Tuberkulose im Erwachsenenalter.

In Germany tuberculosis is a rare disease and usually well treatable. Worldwide it is one of the most common infectious diseases with approximately 10 million new cases every year. Even with low incidences in Germany, tuberculosis is an important differential diagnosis especially due to international developments and migration movements. With a decreasing experience there's a continuous demand on accurate and up-to-date information. This guideline covers all aspects of microbiological diagnostics, basic principles of standard therapy, treatment of extrapulmonary tuberculosis, management of side effects, special features of diagnosis and treatment of resistant tuberculosis, and treatment in TB-HIV coinfection. Also, it explains when treatment in specialized centers is required, aspects of care and legal regulations and the diagnosis and preventive therapy of latent tuberculosis infection. The update of the S2k guideline "Tuberculosis in Adults" is intended to serve as a guideline for prevention, diagnosis, and treatment of tuberculosis for all those involved in tuberculosis care and to help meet the current challenges in dealing with tuberculosis in Germany.

Prevalence and Molecular Characterization of Mycobacterium bovis in Slaughtered Cattle Carcasses in Burkina Faso; West Africa.

This cross-sectional study was conducted at the slaughterhouses/slabs of Oudalan and Ouagadougou in Burkina Faso, between August and September 2013. It aimed at determining the prevalence of bovine tuberculosis (bTB) suggestive lesions in slaughtered cattle carcasses and to identify and characterize the mycobacteria isolated from these lesions. A thorough postmortem examination was conducted on carcasses of a total of 2165 randomly selected cattle. The overall prevalence of bTB suggestive lesions was 2.7% (58/2165; 95% CI 2.1-3.5%). Due to the low number of positive samples, data were descriptively presented. The lesions were either observed localized in one or a few organs or generalized (i.e., miliary bTB) in 96.6% (n = 57) and 3.4% (n = 2), respectively. The identified mycobacteria were M. bovis (44.4%, n = 20), M. fortuitum (8.9%, n = 4), M. elephantis (6.7%, n = 3), M. brumae (4.4%, n = 2), M. avium (2.2%, n = 1), M. asiaticum (2.2%, n = 1), M. terrae (2.2%, n = 1), and unknown non-tuberculous mycobacteria (NTM) (11.1%, n = 5). Moreover, eight mixed cultures with more than one Mycobacterium species growing were also observed, of which three were M. bovis and M. fortuitum and three were M. bovis and M. elephantis. In conclusion, M. bovis is the predominant causative agent of mycobacterial infections in the study area. Our study has identified a base to broaden the epidemiological knowledge on zoonotic transmission of mycobacteria in Burkina Faso by future studies investigating further samples from humans and animals, including wild animals employing molecular techniques.

[Patients with lung disease caused by non-tuberculous mycobacteria in Germany: a trans-sectoral patient-oriented care concept]. / Versorgung von Patienten/innen mit einer durch nichttuberkulöse Mykobakterien verursachten Lungenerkrankung in Deutschland ­ ein transsektorales patientenorientiertes Versorgungskonzept.

INTRODUCTION: Management of patients with lung disease caused by non-tuberculous mycobacteria (NTM-LD) in Germany is currently characterized by delayed diagnosis, frequently poor prognosis and high follow-up costs. Mainly due to an increased number of hospitalizations, the SHI-relevant direct costs (€ 9,093.20 patient/year) are higher compared to typical underlying diseases (e.g. asthma: € 706.00 patient/year). This less than optimal NTM care is mainly caused by lack of awareness of the disease at primary care and out-patient specialist care level, largely absent structured referral structures and limited communication between specialists out of hospital with specialized NTM clinics. Lack of incentives to support these communication pathways is part of the problem. Sufficient, appropriate and economically sustainable care is hampered by poor adherence to treatment recommendations. METHODS: For the development of the NTM care concept, relevant professional societies and patient organizations were interviewed about the care situation. Thereafter, 20 NTM-LD patients, 5 residential pulmonologists and 8 experts were interviewed in an explorative qualitative interview to determine the current patient pathway. Based on the findings, the NTM care concept was developed in an advisory board by the authors. RESULTS: Regional management centers should concentrate specific expertise and ensure quality of care through routine consultation and involvement in diagnosis, decision-making on treatment necessity, initiation of therapy, follow-up examinations, and determination of the therapy success, as well as adequate follow-up of patients. The referring pulmonologist should continue to provide case-specific therapy support close to the patient's home in preferred shared-care concept. The establishment of clear referral structures and case identification criteria will help residential physicians to include patients at risk in the NTM-care system early. Patients and pulmonologists without specific expertise need to be made aware of the care pathway and severity of NTM-LD. CONCLUSION: The increased morbidity and mortality of NTM-LD patients must be addressed with patient-oriented, interdisciplinary and trans-sectoral care concept. An NTM care system with clear treatment procedures and referral structures is proposed for a nationwide pilot project.

Hodgkin Lymphoma after Disseminated Mycobacterium genavense Infection, Germany.

Mycobacterium genavense infection, a rare nontuberculous mycobacteria infection, occurs in heavily immunocompromised patients (i.e., those with advanced HIV disease, genetic disorders, or acquired immunologic disorders and those undergoing immunosuppressive therapy). We report a case of disseminated M. genavense infection preceding Hodgkin lymphoma in a patient without obvious risk factors for this infection.

Mycobacterium tuberculosis Complex Lineage 3 as Causative Agent of Pulmonary Tuberculosis, Eastern Sudan1.

Pathogen-based factors associated with tuberculosis (TB) in eastern Sudan are not well defined. We investigated genetic diversity, drug resistance, and possible transmission clusters of Mycobacterium tuberculosis complex (MTBC) strains by using a genomic epidemiology approach. We collected 383 sputum specimens at 3 hospitals in 2014 and 2016 from patients with symptoms suggestive of TB; of these, 171 grew MTBC strains. Whole-genome sequencing could be performed on 166 MTBC strains; phylogenetic classification revealed that most (73.4%; n = 122) belonged to lineage 3 (L3). Genome-based cluster analysis showed that 76 strains (45.9%) were grouped into 29 molecular clusters, comprising 2-8 strains/patients. Of the strains investigated, 9.0% (15/166) were multidrug resistant (MDR); 10 MDR MTBC strains were linked to 1 large MDR transmission network. Our findings indicate that L3 strains are the main causative agent of TB in eastern Sudan; MDR TB is caused mainly by transmission of MDR L3 strains.

Disseminated Mycosis by Arthrocladium fulminans Jeopardizing a Patient with GATA2 Deficiency.

GATA2 deficiency is characterized by monocytopenia, deficiency of dendritic cells, and a variable degree of lymphocytopenia affecting B cells and NK cells, leading to an enhanced risk of mycobacterial, viral, and fungal infections. Here we present a patient with a heterozygous intronic GATA2 mutation who acquired a fatal disseminated mycosis due to the black yeast-like fungus Arthrocladium fulminans following an infection with Mycobacterium sherrisii. This case illustrates that in patients with severe uncommon infections, immunodeficiency syndromes must be ruled out.

Longitudinal Outbreak of Multidrug-Resistant Tuberculosis in a Hospital Setting, Serbia.

A retrospective population-based molecular epidemiologic study of multidrug-resistant Mycobacterium tuberculosis complex strains in Serbia (2008-2014) revealed an outbreak of TUR genotype strains in a psychiatric hospital starting around 1990. Drug unavailability, poor infection control, and schizophrenia likely fueled acquisition of additional resistance and bacterial fitness-related mutations over 2 decades.

Mycobacterium Growth Inhibition Assay of Human Alveolar Macrophages as a Correlate of Immune Protection Following Mycobacterium bovis Bacille Calmette-Guérin Vaccination.

BACKGROUND: In order to eliminate tuberculosis (TB), an effective vaccine is urgently needed to prevent infection with Mycobacterium tuberculosis. A key obstacle for the development of novel TB vaccines is the lack of surrogate markers for immune protection against M. tuberculosis. METHODS: We investigated growth rates of M. tuberculosis in the mycobacterial growth inhibition assay (MGIA) as a marker for mycobacterial growth control of human bronchoalveolar lavage (BALC) and peripheral blood mononuclear cells (PBMC) before and after vaccination with Mycobacterium bovis Bacille Calmette-Guérin (BCG) of healthy adult volunteers. RESULTS: Vaccination induced a positive response (p < 0.001) to purified protein derivate (PPD) in 58.8% of the individuals in an interferon-γ release assay-ELISpot. Intraindividual evaluation of the MGIA growth rates before and after M. bovis BCG-vaccination revealed no significant difference in time to culture positivity before and after vaccination in BALC (p = 0.604) and PBMC (p = 0.199). The magnitude of the PPD-response induced by M. bovis BCG-vaccination did not correlate with growth control in BALC and PBMC (correlation = 0.468, 95% CI: -0.016 to 0.775). CONCLUSION: In conclusion, M. bovis BCG-vaccination-induced mycobacterial-specific cytokine immune response does not result in functional immune control against M. tuberculosis in the MGIA.

Smear Microscopy for Diagnosis of Pulmonary Tuberculosis in Eastern Sudan.

BACKGROUND: In Sudan, tuberculosis diagnosis largely relies on clinical symptoms and smear microscopy as in many other low- and middle-income countries. The aim of this study was to investigate the positive predictive value of a positive sputum smear in patients investigated for pulmonary tuberculosis in Eastern Sudan. METHODS: Two sputum samples from patients presenting with symptoms suggestive of tuberculosis were investigated using direct Ziehl-Neelsen (ZN) staining and light microscopy between June to October 2014 and January to July 2016. If one of the samples was smear positive, both samples were pooled, stored at -20°C, and sent to the National Reference Laboratory (NRL), Germany. Following decontamination, samples underwent repeat microscopy and culture. Culture negative/contaminated samples were investigated using polymerase chain reaction (PCR). RESULTS: A total of 383 samples were investigated. Repeat microscopy categorized 123 (32.1%) as negative, among which 31 were culture positive. This increased to 80 when PCR and culture results were considered together. A total of 196 samples were culture positive, of which 171 (87.3%), 14 (7.1%), and 11 (5.6%) were M. tuberculosis, M. intracellulare, and mixed species. Overall, 15.6% (57/365) of the samples had no evidence of M. tuberculosis, resulting in a positive predictive value of 84.4%. CONCLUSIONS: There was a discordance between the results of smear microscopy performed at local laboratories in the Sudan and at the NRL, Germany; besides, a considerable number of samples had no evidence of M. tuberculosis. Improved quality control for smear microscopy and more specific diagnostics are crucial to avoid possible overtreatment.

Microbiologic Outcome of Interventions Against Mycobacterium avium Complex Pulmonary Disease: A Systematic Review.

OBJECTIVE: Pulmonary disease (PD) caused by Mycobacterium avium complex (MAC) is increasing worldwide. We conducted a systematic review of studies that include microbiologic outcomes to evaluate current macrolide-based treatment regimens. METHODS: We searched literature published before April 2017 by using the MEDLINE, Cochrane, and Embase databases. Risk of bias in randomized trials was assessed using the Cochrane tool. RESULTS: We retrieved 333 citations and evaluated 42 studies including 2,748 patients: 18 studies were retrospective chart reviews, 18 were prospective, and six were randomized. The weighted average proportion of sputum culture conversions in macrolide-containing regimens after subtracting posttreatment microbiologic recurrences was 52.3% (95% CI, 44.7%-59.9%). Using the triple-drug regimens recommended by the American Thoracic Society (ATS) achieved treatment success in 61.4% (95% CI, 49.7%-72.5%), which further increased to 65.7% (95% CI, 53.3%-77.4%) when drugs were taken for at least 1 year by patients who were macrolide susceptible and had previously untreated MAC. The overall risk of bias was low in five of the six randomized trials. However, selective outcome reporting because of a posteriori exclusion of initially included patients (14.0%), uncompleted treatment (17.6%), and inconsistent use of outcome parameters (17 definitions of treatment success) hampered the comparison of nonrandomized trials. CONCLUSIONS: To date, randomized studies on treatment outcome in patients with MAC PD are scarce. Long-term treatments with ATS-recommended regimens for patients who are macrolide susceptible are superior to other macrolide-based therapies. A standardized definition of treatment success and genotypic distinction between reinfection and relapse by means of pretreatment and posttreatment identification of MAC species in cases of microbiologic recurrences may help to optimize evaluation of treatment regimens in the future.

Integration of molecular typing results into tuberculosis surveillance in Germany-A pilot study.

An integrated molecular surveillance for tuberculosis (TB) improves the understanding of ongoing TB transmission by combining molecular typing and epidemiological data. However, the implementation of an integrated molecular surveillance for TB is complex and requires thoughtful consideration of feasibility, demand, public health benefits and legal issues. We aimed to pilot the integration of molecular typing results between 2008 and 2010 in the German Federal State of Baden-Württemberg (population 10.88 Million) as preparation for a nationwide implementation. Culture positive TB cases were typed by IS6110 DNA fingerprinting and results were integrated into routine notification data. Demographic and clinical characteristics of cases and clusters were described and new epidemiological links detected after integrating typing data were calculated. Furthermore, a cross-sectional survey was performed among local public health offices to evaluate their perception and experiences. Overall, typing results were available for 83% of notified culture positive TB cases, out of which 25% were clustered. Age <15 years (OR = 4.96, 95% CI: 1.69-14.55) and being born in Germany (OR = 2.01, 95% CI: 1.44-2.80) were associated with clustering. At cluster level, molecular typing information allowed the identification of previously unknown epidemiological links in 11% of the clusters. In 59% of the clusters it was not possible to identify any epidemiological link. Clusters extending over different counties were less likely to have epidemiological links identified among their cases (OR = 11.53, 95% CI: 3.48-98.23). The majority of local public health offices found molecular typing useful for their work. Our study illustrates the feasibility of integrating typing data into the German TB notification system and depicts its added public health value as complementary strategy in TB surveillance, especially to uncover transmission events among geographically separated TB patients. It also emphasizes that special efforts are required to strengthen the communication between local public health offices in different counties to enhance TB control.

[Tuberculosis Guideline for Adults - Guideline for Diagnosis and Treatment of Tuberculosis including LTBI Testing and Treatment of the German Central Committee (DZK) and the German Respiratory Society (DGP)]. / S2k-Leitlinie: Tuberkulose im Erwachsenenalter.

Since 2015 a significant increase in tuberculosis cases is notified in Germany, mostly due to rising numbers of migrants connected to the recent refugee crisis. Because of the low incidence in previous years, knowledge on tuberculosis is more and more limited to specialized centers. However, lung specialist and healthcare workers of other fields have contact to an increasing number of tuberculosis patients. In this situation, guidance for the management of standard therapy and especially for uncommon situations will be essential. This new guideline on tuberculosis in adults gives recommendations on diagnosis, treatment, prevention and prophylaxis. It provides a comprehensive overview over the current knowledge, adapted to the specific situation in Germany. The German Central Committee against Tuberculosis (DZK e. V.) realized this guideline on behalf of the German Respiratory Society (DGP). A specific guideline for tuberculosis in the pediatrics field will be published separately. Compared to the former recommendations of the year 2012, microbiological diagnostics and therapeutic drug management were given own sections. Chapters about the treatment of drug-resistant tuberculosis, tuberculosis in people living with HIV and pharmacological management were extended. This revised guideline aims to be a useful tool for practitioners and other health care providers to deal with the recent challenges of tuberculosis treatment in Germany.

Microbiological and Clinical Outcomes of Treating Non-Mycobacterium Avium Complex Nontuberculous Mycobacterial Pulmonary Disease: A Systematic Review and Meta-Analysis.

BACKGROUND: Pulmonary disease caused by nontuberculous mycobacteria (NTM) is steadily increasing worldwide. METHODS: A systematic review of non-Mycobacterium avium complex studies published prior to October 2016 was conducted with respect to microbiological and clinical outcomes of current treatment regimens. RESULTS: We retrieved 352 citations, which yielded 24 studies eligible for evaluation. Sixteen studies were retrospective chart reviews, three studies were prospective, and only five studies were randomized. The weighted average proportion of sputum culture conversion (SCC) after subtracting posttreatment relapses for patients with M abscessus was 41.2% (95% CI, 28.6%-54.5%) but was 69.8% (95% CI, 41.0%-91.9%) with subspecies M massiliense in macrolide-containing regimens, 80.2% (95% CI, 58.4%-95.2%) in patients with M kansasii, 32.0% (95% CI, 16.5%-49.8%) for M xenopi (MX) and 54.4% (95% CI, 34.7%-73.4%) for M malmoense. SCCs in the total of 55 patients who underwent lung resection and had MX or M abscessus was high at 75.9%. The risk of bias was low in four of five randomized studies. However, heterogeneous use of outcome parameters (eight definitions of "relapse," eight of "treatment success," and four of "cure") hampered comparison of nonrandomized studies as well as producing possible bias by a posteriori exclusion (13.3%) and uncompleted treatment of participants (25.3%). CONCLUSIONS: As a sustained microbiological response without surgery is unsatisfactory in treating M abscessus, MX, and M malmoense, functional and quality of life aspects should be given more emphasis in the individual evaluation of treatment outcome. Further, properly planned studies with sufficient power are needed, as are new drugs or better-tolerated application of current antibiotics, or both.

A joint cross-border investigation of a cluster of multidrug-resistant tuberculosis in Austria, Romania and Germany in 2014 using classic, genotyping and whole genome sequencing methods: lessons learnt.

Molecular surveillance of multidrug-resistant tuberculosis (MDR-TB) using 24-loci MIRU-VNTR in the European Union suggests the occurrence of international transmission. In early 2014, Austria detected a molecular MDR-TB cluster of five isolates. Links to Romania and Germany prompted the three countries to investigate possible cross-border MDR-TB transmission jointly. We searched genotyping databases, genotyped additional isolates from Romania, used whole genome sequencing (WGS) to infer putative transmission links, and investigated pairwise epidemiological links and patient mobility. Ten isolates from 10 patients shared the same 24-loci MIRU-VNTR pattern. Within this cluster, WGS defined two subgroups of four patients each. The first comprised an MDR-TB patient from Romania who had sought medical care in Austria and two patients from Austria. The second comprised patients, two of them epidemiologically linked, who lived in three different countries but had the same city of provenance in Romania. Our findings strongly suggested that the two cases in Austrian citizens resulted from a newly introduced MDR-TB strain, followed by domestic transmission. For the other cases, transmission probably occurred in the same city of provenance. To prevent further MDR-TB transmission, we need to ensure universal access to early and adequate therapy and collaborate closely in tuberculosis care beyond administrative borders.

Detection of transrenal DNA for the diagnosis of pulmonary tuberculosis and treatment monitoring.

PURPOSE: Molecular diagnostics of patients with MTB tuberculosis from urine samples. METHODS: We developed a new molecular assay based on the detection of M. tuberculosis-specific transrenal DNA (trDNA) and tested it for the diagnosis of active tuberculosis at the initiation of anti-tuberculosis therapy and during treatment follow-up. RESULTS: The overall sensitivity of trDNA was 96 and 100% when smear-microscopy and trDNA was combined. In a subset of TB treatment naïve patients (n = 11) sensitivity and specificity of trDNA was 64 and 100%, respectively. For this subset of patients the sensitivity was 91% when smear-microscopy and trDNA diagnosis were combined. After treatment initiation, trDNA showed a significant reduction in concentration over time reaching undetectable trDNA values at week 12 in 9 of 11 accessible patients (82%). Kinetics in treatment-naïve patients showed low base-line trDNA levels, which increased to maximal trDNA levels within one week indicating bactericidal activity of anti-tuberculosis drugs after the initiation of effective therapy. Maximal trDNA levels correlated positively with a radiological score, suggesting that the process of DNA excretion may reflect the extent of pulmonary disease. Matched samples showed an inverse correlation between the time to positivity of solid culture with maximum trDNA levels as well as the expected positive correlation between smear grade and maximum trDNA values. CONCLUSION: The detection of M. tuberculosis trDNA from urine specimen is a promising method for the diagnosis tuberculosis. The assay may be a candidate diagnostic tool for patients with paucibacillary and extrapulmonary disease, as method to assess treatment responses and could be helpful to diagnose tuberculosis in children.

Raman spectroscopic identification of Mycobacterium tuberculosis.

In this study, Raman microspectroscopy has been utilized to identify mycobacteria to the species level. Because of the slow growth of mycobacteria, the per se cultivation-independent Raman microspectroscopy emerges as a perfect tool for a rapid on-the-spot mycobacterial diagnostic test. Special focus was laid upon the identification of Mycobacterium tuberculosis complex (MTC) strains, as the main causative agent of pulmonary tuberculosis worldwide, and the differentiation between pathogenic and commensal nontuberculous mycobacteria (NTM). Overall the proposed model considers 26 different mycobacteria species as well as antibiotic susceptible and resistant strains. More than 8800 Raman spectra of single bacterial cells constituted a spectral library, which was the foundation for a two-level classification system including three support vector machines. Our model allowed the discrimination of MTC samples in an independent validation dataset with an accuracy of 94% and could serve as a basis to further improve Raman microscopy as a first-line diagnostic point-of-care tool for the confirmation of tuberculosis disease.

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.

BACKGROUND: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. METHODS: To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. RESULTS: dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. CONCLUSIONS: TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.